The effect of casein glycomacropeptide versus free synthetic amino acids for early treatment of phenylketonuria in a mice model

Kirsten K Ahring; Frederik Dagnæs-Hansen; Annemarie Brüel; Mette Christensen; Erik Jensen; Thomas G Jensen; Mogens Johannsen; Karen S Johansen; Allan M Lund; Jesper G Madsen; Karen Brøndum-Nielsen; Michael Pedersen; Lambert K Sørensen; Mads Kjolby; Lisbeth B Møller

doi:10.1371/journal.pone.0261150

. 2022 Jan 11;17(1):e0261150. doi: 10.1371/journal.pone.0261150

The effect of casein glycomacropeptide versus free synthetic amino acids for early treatment of phenylketonuria in a mice model

Kirsten K Ahring ^1,^*, Frederik Dagnæs-Hansen ², Annemarie Brüel ², Mette Christensen ³, Erik Jensen ⁴, Thomas G Jensen ², Mogens Johannsen ⁵, Karen S Johansen ², Allan M Lund ³, Jesper G Madsen ², Karen Brøndum-Nielsen ¹, Michael Pedersen ⁶, Lambert K Sørensen ⁵, Mads Kjolby ^2,^7,⁸, Lisbeth B Møller ⁹

Editor: Aneta Agnieszka Koronowicz¹⁰

¹Departments of Paediatrics and Clinical Genetics, PKU Clinic, Kennedy Center, Copenhagen University Hospital, Rigshospitalet, Denmark

²Department of Biomedicine, Health, Aarhus University, Aarhus, Denmark

³Departments of Paediatrics and Clinical Genetics, Centre for Inherited Metabolic Diseases, Copenhagen University Hospital, Rigshospitalet, Denmark

⁴Arla Foods Ingredients Group P/S, Viby J, Denmark

⁵Department of Forensic Medicine, Aarhus University, Skejby, Aarhus, Denmark

⁶Comparative Medicine Lab, Aarhus University Hospital, Aarhus, Denmark

⁷Steno Diabetes Center Aarhus, Aarhus University Hospital, Aarhus, Denmark

⁸Department of Clinical Pharmacology, Aarhus University Hospital, Aarhus, Denmark

⁹Department of Clinical Genetics, Kennedy Center, Copenhagen University Hospital, Rigshospitalet, Denmark

¹⁰University of Agriculture in Krakow, POLAND

Competing Interests: Erik Jensen was the industrial supervisor for the above described PhD project, and his mandatory obligation was to contribute with inputs to study design, formulation of test diets and study manuscript according to agreements and ethical standard. However, Erik Jensen did not have the final decision about these issues and this affiliation does not alter our adherence to PLOS ONE policies on sharing data and materials. Erik Jensen has sent a written signed statement regarding this (separate file).

^✉

* E-mail: kirsten.kiaer.ahring@regionh.dk

Roles

Kirsten K Ahring: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Writing – original draft

Frederik Dagnæs-Hansen: Conceptualization, Investigation, Project administration, Supervision, Writing – review & editing

Annemarie Brüel: Conceptualization, Data curation, Formal analysis, Supervision, Writing – review & editing

Mette Christensen: Formal analysis, Methodology, Validation, Writing – review & editing

Erik Jensen: Conceptualization, Formal analysis, Funding acquisition, Project administration, Supervision, Writing – review & editing

Thomas G Jensen: Conceptualization, Resources, Writing – review & editing

Mogens Johannsen: Data curation, Formal analysis, Methodology, Supervision, Writing – review & editing

Karen S Johansen: Data curation, Investigation, Supervision, Writing – review & editing

Allan M Lund: Project administration, Supervision, Writing – review & editing

Jesper G Madsen: Investigation, Writing – review & editing

Karen Brøndum-Nielsen: Conceptualization, Supervision, Writing – review & editing

Michael Pedersen: Conceptualization, Formal analysis, Investigation, Methodology, Supervision, Writing – review & editing

Lambert K Sørensen: Formal analysis, Investigation, Methodology, Supervision, Validation, Writing – review & editing

Mads Kjolby: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Supervision, Writing – review & editing

Lisbeth B Møller: Conceptualization, Formal analysis, Methodology, Project administration, Supervision, Writing – review & editing

Aneta Agnieszka Koronowicz: Editor

PMCID: PMC8751992 PMID: 35015767

Abstract

Introduction

Management of phenylketonuria (PKU) is mainly achieved through dietary control with limited intake of phenylalanine (Phe) from food, supplemented with low protein (LP) food and a mixture of free synthetic (FS) amino acids (AA) (FSAA). Casein glycomacropeptide (CGMP) is a natural peptide released in whey during cheese making by the action of the enzyme chymosin. Because CGMP in its pure form does not contain Phe, it is nutritionally suitable as a supplement in the diet for PKU when enriched with specific AAs. Lacprodan^® CGMP-20 (= CGMP) used in this study contained only trace amounts of Phe due to minor presence of other proteins/peptides.

Objective

The aims were to address the following questions in a classical PKU mouse model: Study 1, off diet: Can pure CGMP or CGMP supplemented with Large Neutral Amino Acids (LNAA) as a supplement to normal diet significantly lower the content of Phe in the brain compared to a control group on normal diet, and does supplementation of selected LNAA results in significant lower brain Phe level?. Study 2, on diet: Does a combination of CGMP, essential (non-Phe) EAAs and LP diet, provide similar plasma and brain Phe levels, growth and behavioral skills as a formula which alone consist of FSAA, with a similar composition?.

Material and methods

45 female mice homozygous for the Pah^enu2 mutation were treated for 12 weeks in five different groups; G1(N-CGMP), fed on Normal (N) casein diet (75%) in combination with CGMP (25%); G2 (N-CGMP-LNAA), fed on Normal (N) casein diet (75%) in combination with CGMP (19,7%) and selected LNAA (5,3% Leu, Tyr and Trp); G3 (N), fed on normal casein diet (100%); G4 (CGMP-EAA-LP), fed on CGMP (70,4%) in combination with essential AA (19,6%) and LP diet; G5 (FSAA-LP), fed on FSAA (100%) and LP diet. The following parameters were measured during the treatment period: Plasma AA profiles including Phe and Tyr, growth, food and water intake and number of teeth cut. At the end of the treatment period, a body scan (fat and lean body mass) and a behavioral test (Barnes Maze) were performed. Finally, the brains were examined for content of Phe, Tyr, Trp, dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), serotonin (5-HT) and 5-hydroxyindole-acetic acid (5-HIAA), and the bone density and bone mineral content were determined by dual-energy x-ray absorptiometry.

Results

Study 1: Mice off diet supplemented with CGMP (G1 (N-CGMP)) or supplemented with CGMP in combination with LNAA (G2 (N-CGMP-LNAA)) had significantly lower Phe in plasma and in the brain compared to mice fed only casein (G3 (N)). Extra LNAA (Tyr, Trp and Leu) to CGMP did not have any significant impact on Phe levels in the plasma and brain, but an increase in serotonin was measured in the brain of G2 mice compared to G1. Study 2: PKU mice fed with mixture of CGMP and EAA as supplement to LP diet (G4 (CGMP-EAA-LP)) demonstrated lower plasma-Phe levels but similar brain- Phe levels and growth as mice fed on an almost identical combination of FSAA (G5 (FSAA-LP)).

Conclusion

CGMP can be a relevant supplement for the treatment of PKU.

Introduction

Phenylketonuria (PKU) (OMIM 261600) is an inherited, autosomal recessive metabolic disorder, caused by reduced conversion of phenylalanine (Phe) to tyrosine (Tyr), due to deficient phenylalanine hydroxylase (PAH) activity, resulting in increased blood Phe levels [1–4]. Without treatment PKU results in severe mental retardation, microcephaly, epilepsy and other neurological symptoms [5]. Also, decreased bone mineral density (BMD) have been reported in patients with PKU [6]. Lifelong adherence to low protein (LP) diet supplemented with free synthetic (FS) amino acids (AA) (FSAA) is recommended, but compliance is often poor, partly due to the taste of the FSAA mixtures [7–9]. Therefore, new treatment options are explored.

Casein glycomacropeptide (CGMP) is a natural 64-amino acid peptide released in whey during cheese making by the action of the enzyme chymosin [10]. CGMP in its pure form does not contain Phe, and this makes it suitable as supplement for patients with PKU when supplemented with adequate amounts of essential amino acids (EAA), tyrosine (Tyr), tryptophan (Trp), histidine (His), arginine (Arg), methionine (Met), lysine (Lys), and leucine (Leu). CGMP contains 2- to 3-fold of isoleucine (Ile), valine (Val) and threonine (Thr) compared to the concentrations found in other dietary proteins [11]. Lacprodan^® CGMP-20 (= CGMP) used in this study contained trace amounts of Phe due to minor presence of other proteins/peptides. Lacprodan^® CGMP-20 will here be referred to as CGMP.

In recent years, trials for evaluation of safety, acceptability and efficacy in mice and humans have demonstrated that CGMP, supplemented with EAA to make it nutritionally adequate, is a safe alternative to conventional treatment with FSAA mixtures [12–17].

A large number of studies have demonstrated that LNAAs, and especially Leu, Tyr and Trp have the ability to reduce Phe entering the brain [18–21]. As CGMP contains large amount of Trp, CGMP might potentially hamper Phe entering the brain.

Furthermore, since CGMP is a 64-amino acid natural peptide and absorbed as di- and tri-peptides as well as free AAs from the gut and subsequently from blood to the brain, this can potentially lead to different concentrations of AA and metabolites in blood and brain compared to absorption of FSAA.

The aims of this study were to address the following questions: Study 1, off diet: Can pure CGMP or CGMP supplemented with the LNAAs; Tyr, Leu and Trp significantly lower the content of Phe in the brain? Does the supplementation of extra LNAA results in significantly lower brain Phe level compared to supplementation of CGMP alone? Study 2, on diet: Does a combination of CGMP, EAAs and LP diet, provide similar plasma and brain Phe levels, growth and behavioral skills as a formula with a similar combination of pure FSAA?

This study is to our knowledge the first study to compare almost identical combinations of FSAA and CGMP in a PKU mouse model. Furthermore, it is noteworthy, that the length of this study is 12 weeks diet intervention, which is about twice as long as similar studies [12, 22].

Material and methods

Animals

The facilities and protocols used in this study were approved by The Danish Experimental Animal Inspectorate (License no. 2013-15-2934-00878). Experimental animals were produced by breeding B6;BTBR mice homozygous for the Pah^enu2 mutation to yield homozygous PKU mice [23]. This mouse strain, the Pah^enu2, was a mix between the black and tan, brachyury (BTBR) mouse and the C57Bl/6J (B6) mouse. Further genotyping of 6 randomly chosen animals from the mouse strain showed that they were between 93%-95% identical with B6 compared to BTBR, which confirms the almost identical behavior with B6 and equally difference with BTBR [22, 24]. The breeding animals were maintained on Phe-free semi synthetic diet (Harlan Laboratories), also during pregnancy and weaning period. As the diet was free of Phe, the drinking water was supplemented with Phe (Sigma-Aldrich Chemie) to a final concentration of 563 mg/l during that period. Mice were housed in open cages in groups of 2–5 mice. Cages were with Tapvei 2HV bedding (L: 37 cm × W: 21 cm × H: 15 cm; a maximum of 5 mice per cage). The facility was temperature-controlled at 22 °C on a 12-12-h light-dark cycle, and the mice were fed ad libitum with the different diets and had free access to water. Environmental enrichment included house, Tapvei s-bricks and Ancare NES3600 nestlets (Brogaarden). One mouse (control group G3 (N)) died after week 5. During the experiment all the animals were weighted every week. The protocol can be found here [25].

The mouse strain used in this study was obtained by breeding mice homozygous for the PAH^enu2 mutation, and unfortunately, no wild-type mouse of this strain is available. However, as the purpose of the present study was to investigate the effect of different diets, it is still possible to achieve even without a normal control. The group of mice on normal casein diet G3(N) is used as a control for the different diets used.

The research diets

The diets were made by Research Diets according to recipes supplied by the investigator, irradiated and stored at 5 °C until use. The AA profiles were made to meet the requirements for mice, which included 5 g/100 g protein equivalent (PE) supplementation of Arg and Met (4.5 g/100 g PE) [26, 27]. All the diets were isocaloric with the AA/protein source being the only source of variation. The diet for group 1 (G1 (N-CGMP)) contained 25% CGMP and 75% casein (Normal diet, N). The diet for group 2 (G2 (N-CGMP-LNAA)) was similar to G1 but supplemented with Trp, Tyr and Leu to test the potential blocking effect in the gut and at the blood brain barrier. The diet for group 4 (G4 (CGMP-EAA-LP)) consisted of CGMP supplemented with EAA to meet the requirements for mice [26, 27]. Group 5 (G5 (FSAA-LP)) was on LP diet supplemented with FSAA and had a similar AA profile as G4 (CGMP-EAA-LP). The diet for group 3 (G3 (N)) is a complete “normal diet (N), consisting of casein (MIPRODAN^® 30), sucrose, corn starch, corn oil, cellulose, mineral- and vitamin-mix. Basically, the nutritional value is the same in all diets. Phe was added to the drinking water in G4 (CGMP-EAA-LP) and G5 (FSAA-LP) (final concentration 563 mg/l) to prevent Phe deficiency since the CGMP and AA mixture served as the sole protein source for these mice. The mice in G1 (N-CGMP), G2 (N-CGMP-LNAA) and G3 (N) drank normal water. The detailed contents of the diets are shown in Tables 1 and 2. Food and water intake were calculated as the mean of intake /week /mice by dividing the total amount of intake in each cage per week (week 1–11), divided by the number of animals per cage.

Table 1. Composition of the experimental diet for the 5 groups (G1, G2, G3, G4, G5) and content of AA in CGMP-20 (g AA/100 g protein).

	Ingrediens	OFF DIET			ON DIET
	Group	G1 (N-CGMP)	G2 (N-CGMP-LNAA)	G3 (N)	G4 (CGMP-EAA-LP)	G5 (FSAA-LP)
		g	g	g	g	g
	Casein (Miprodan^® 30)	155.25	155.25	207.00	0	0
	Sucrose	458.63	458.75	465.98	454.75	471.68
	Corn Starch	150.00	150.00	150.00	150.00	150.00
	Corn Oil	100.00	100.00	100.00	100.00	100.00
	Cellulose	30.00	30.00	30.00	30.00	30.00
	Mineral Mix. AIN-76 (170915)	35.00	35.00	35.00	35.00	35.00
	Vitamin Mix. standard	10.00	10.00	10.00	10.00	10.00
	Ethoxyquin. antioxidant	0.02	0.02	0.02	0.02	0.02
	Choline bitartrate	2.00	2.00	2.00	2.00	2.00
Lacprodan^® CGMP-20 g AA/ 100 g protein		59.10	46.63		153.70
6.4	Ala					8.50
0.3	Arg				11.42	11.86
9.2	Aspartic Acid					11.98
0.08	Cys					0.15
21.1	Glutamic Acid					26.30
1.2	Glycine					1.52
0.2	His				4.41	4.57
11.5	Ile					14.38
2.5	Leu		1.36		15.14	18.24
6.4	Lys				3.46	11.86
2	Met				5.31	8.20
0.2	Phe				0.45	0.65
12.6	Pro					16.31
8.5	Ser					10.37
18.1	Thr					23.42
0.04	Trp		3.43		3.66	3.66
0.06	Tyr		7.56		16.36	16.41
9.5	Val				2.20	13.85
109.88	TOTAL	1000.00	1000.00	1000.00	1000.00	1000.00

Open in a new tab

The composition is based on the following recommendation: WHO Technical Report Series. Protein and amino acid requirements in human nutrition. 2007 [28]. See also Table 2.

* G1-G5 refers to groups 1–5. In addition to CGMP, G2 and G4 received additional AA in form of FSAA (% indicates fraction of AA received in the form of FSAA): Off diet (G2): Leu: 53.75%, Trp: 99.42% and Tyr: 99.6%. On diet (G4): Arg: 96.25%, His: 93.44%, Leu: 79.76%, Lys: 30.42%, Met: 64.60%, Trp: 98.36%, Tyr: 99.44% and Val: 15.56% G4 and G5 receive in addition Phe from the drinking water (0,563mg Phe/ml). Each animal drank on average 30.3 ml (G4 ~17mg Phe/week) and 38.2 ml (G5~ 22mg Phe/week). In comparison each animal ate an average of 14.6g /week (G4) and 15.4g/week (G5) respectively.

Table 2. Analysis of diets G1-G5.

The diets G4 (CGMP-EAA-LP) and G5 (FSAA-LP) receive in addition Phe from the drinking water (0,563mg Phe/ml).

Ingredients	OFF DIET			ON DIET
Group	G1 (N-CGMP)	G2 (N-CGMP-LNAA)	G3 (N)	G4 (CGMP-EAA-LP)	G5 (FSAA-LP)
Protein (%)	18.0	17.9	18.2	17.2	15.6
Carbohydrate (%)	63.2	63.5	64.4	64.2	68.3
Fat (%)	9.2	9.3	9	9.1	10.1
Minerals (%)	3.3	3.3	3.2	3.5	2.4
Moisture (%)	6.3	6	5.3	6.1	3.6
g AA/100g
Ala	0.73	0.68	0.59	0.81	1.02
Arg	0.51	0.52	0.70	1.14	1.1
Aspartic Acid	1.42	1.35	1.37	1.12	1.07
Cys	0.09	0.08	0.10	0.04	0.03
Glutamic Acid	4.05	3.86	4.23	2.6	2.59
Glycine	0.32	0.31	0.36	0.15	0.15
His	0.41	0.42	0.56	0.27	0.23
Ile	1.23	1.13	0.98	1.37	1.43
Leu	1.43	1.53	1.81	1.72	1.77
Lys	1.40	1.36	1.56	1.18	0.92
Met	0.48	0.48	0.52	0.75	0.73
Phe	0.74	0.74	1.01	0.06	0.06
Pro	2.15	2.01	2.12	1.57	1.58
Ser	1.19	1.12	1.10	0.99	0.96
Thr	1.46	1.30	0.83	2.22	2.35
Trp	0.16	0.44	0.21	0.3	0.3
Tyr	0.67	1.33	0.94	1.41	1.44
Val	1.33	1.25	1.25	1.36	1.26
TOTAL	19.77	19.91	20.24	19.06	18.99
Phe in water	No	No	No	Yes	Yes

Open in a new tab

Each animal drank on average 30.3 ml (G4 ~17mg Phe/week) and 38.2 ml (G5~ 22mg Phe/week). In comparison each animal ate an average of 14.6g /week (G4) and 15.4g/week (G5) respectively.

Weekly Phe and Tyr analyses

A blood drop was collected on filter paper (DBS) Whatman FTA/FTA Elute cards (Whatman, GE Healthcare Europe) every week (week 1–12), except for week 6 and 12. At week 6 and 12 bloods for total AA profile analysis were taken. Phe and Tyr content in a punch from the filter paper blood sample was analysed by High Performance Liquid Chromatography (HPLC)-Tandem Mass Spectrometry (TMS) at Statens Serum Institute, Denmark, according to standard protocols.

AA profile analysis in plasma

Blood samples were taken for AA profile analysis after 3.5–5.5 hours of fasting by puncture of the mandibular vein at week 6 and 12 during diet treatment. Plasma was isolated by centrifugation at 3000 × g for 10 min at 4 °C and stored at -80 °C prior to analysis. A small pilot study was performed to validate the test.

Analysis of free AA in plasma was carried out using a Waters Acquity Ultra-Performance Liquid Chromatography (UPLC) (Waters) system with an integrated photodiode array detector and the Mass Trak AA Solution Kit (Waters) according to standard protocols with the slight modifications described by Peake et al [29]. All conditions are available upon request.

Body composition analysis

At the end of the feeding period (week 13), the animals were analysed for body composition by magnetic resonance (MR) scanning (EchoMRI-100H).

Behavior performance test (Barne’s maze behavioral test)

After 1 week of acclimatisation (week 13) a behavioural test (Barne’s Maze) was performed to test short term and long-term memory (week 14–15). Behavioural testing were performed between 8 am and 4 pm. The scientist performing the behavioral studies was blinded to the experimental group. The tests were carried out to asses any cognitive impact of the diet upon the test animals’ memory and spatial learning abilities [30]. Tests were performed on a circular table with 20 holes spread around the circumference of the table. One of the 20 holes was designed as an escape hole, allowing the animals to escape the open surface of the table into a container below, thus utilizing the natural instinct of mice to avoid open spaces and seek cover. The remaining holes provided the mice with only shallow cover. Visual clues, consisting of different colors and patterns, were placed on the walls surrounding the table and the relation between the clues and the escape hole remained unchanged throughout the experimental period. The animals were allowed to rest for 1 hour before testing commenced. Each animal was then placed in its own container with bedding and a metal cover. At the beginning of behavioral trials, the animals were placed centrally on the table, to rest under a cover for 1 min. The cover was then removed, and the animal given 3 min to find the escape hole. If the hole was not found within that time, the hole was shown to the animal by the operator. The bottoms of the non-escape holes were randomly switched between trials as well. Animal performance was recorded using AnyMaze (ANY-Maze) and the metrics were: total time on table, distance traveled, mean speed, time spent to locate the escape hole and whether the trial was successful or not, i.e. the animal located the escape hole within 3 min. On day 1 of testing each animal would complete 5 trials and subsequently 4 trials on day 2 to 5. Once the animals had completed these trials, they were given 7 days rest, then put through 4 additional trials on day 11 in order to test long term memory.

Brain and bone removal

After week 15, the animals were placed under anaesthesia using 4% isoflurane in air and euthanized by cervical dislocation. The brain was harvested, transferred to a petri dish on ice and then dissected into six parts; cerebellum, brain stem, hypothalamus, parietal cortex, anterior piriform, cortex and olfactory bulb and the parts were snap frozen in liquid nitrogen separately and stored at −80 °C until analysis. Afterwards, the two femoral bones were dissected out by opening the hip and knee joints, stripped from muscles and soft connective tissue, and frozen in Ringer-lactate in Eppendorf tubes at -20 °C until analysis [31].

Analysis of Phe, Tyr, Trp, neurotransmitters, and their metabolites in the brain regions

Analysis for content of Phe, Tyr, Trp, 5-hydroxyindole-acetic acid (5-HIAA), 3,4-dihydroxyphenylacetic acid (DOPAC), dopamine (DA) and serotonin (5-HT) was performed at Department of Forensic Medicine, Aarhus University Hospital, Denmark. The brain components (2–140 mg) were homogenised using a Precellys tissue homogeniser (Bertin Technologies) in a volume of 1.1 mL cold 78% acetonitrile containing ascorbic acid and stable isotope labelled internal standards. The AA were analysed directly after dilution of the extract. The other substances were cleaned up by solid phase extraction on ion exchange sorbents. The measurements were performed by (UPLC-MS/MS). The UPLC system was a Waters Acquity system that consisted of a binary pump, a flow-through-needle sample manager thermostated at 5±2 °C and a column oven set at 45±2 °C (Waters). The MS/MS was a Waters Xevo TQ-S triple-quadrupole instrument with an electrospray ionisation ion (ESI) source. The separation was performed using a reversed-phase HSS T3 column (1.8 μm, 200 Å, 2.1 mm I.D. × 100 mm) (Waters). The mobile phases A and B consisted of 10% methanol and methanol/acetonitrile (1+1), both acidified with formic acid and acetic acid. A 10 μL volume was injected onto the column running 100% mobile phase A. The mobile phase was changed through a linear gradient to 90% A and 10% B over 4 min. Then the column was washed and conditioned before next injection. The primary ion transitions used for quantification and qualification were m/z 137 > 65 and 91 (DA), m/z 160 > 77 and 132 (5-HT), m/z 192 > 118 and 146 (5-HIAA), m/z 123 > 123 and 95 (DOPAC), m/z 166 > 131 and 103 (Phe), m/z 182 > 136 and 119 (Tyr) and m/z 205 > 118 and 146 (Trp).

Bone examination

The right femora was thawed, cleaned for soft connective tissue, and femoral length determined using a digital caliper. Subsequently, the femora were placed in a peripheral dual-emission x-ray absorptiometry (DEXA) scanner (Sabre XL; Norland Stratec) and scanned using a pixel resolution of 0.1 × 0.1 mm². The bone mineral content (BMC) and the areal bone mineral density (aBMD) of the whole bone were determined with the software provided by the MR system manufacturer. Quality assurance was performed by scans of the two solid-state phantoms provided with the scanner. The coefficient of variation (CV) of mice femoral aBMD was 2.6% (procedure repeated ten times on the same femur).

Statistical analysis

All calculations were performed using the software SPSS 22 (SPSS; IBM) or Microsoft Excel 2010 for Windows. Paired- and unpaired-t-tests, Pearsons correlation test, one and two-way ANOVA performed two-sided at a significance level of α = 0.05.

The experimental design

The experimental design was set to control for three main effects and their interactions: 1) effect of adding CGMP to a high Phe-diet (G1 (N-CGMP)); 2) adding CGMP to a high Phe-diet supplemented with Tyr, Leu and Trp (G2 (N-CGMP-LNAA)); 3) the effect of using CGMP as the main AA source in a LP diet (G4 (CGMP-EAA-LP)) compared to FSAA (G5 (FSAA-LP)). Mice fed at casein diet served as the control (G3(N)). After the weaning period for 28 days, female offspring were randomized to one of the five diets and kept on this diet through young adulthood until end of the experiment, which in total was a period of 15 weeks. The overall design of the study is shown with accompanied timeline in Fig 1.

Results

Results for all 5 groups are presented in the same figures and tables, but statistically compared in sub-groups G1 (N-CGMP), G2 (N-CGMP-LNAA) and G3 (N) versus G3 (N), G4 (CGMP-EAA-LP) and G5 (FSAA-LP) when relevant, otherwise compared between all groups. G3 (N), untreated mice on casein diet, served as a control group.

Food and water

Food intake were significantly higher for G2 (N-CGMP-LNAA) and G5 (FSAA-LP), when separately compared to G3 (N) (p = 0.01). Water: G2 (N-CGMP-LNAA) had an almost 2-fold increased intake of water compared to both G1 (N-CGMP) and G3 (N) (p < 0.01). G3 had the lowest intake of water in total but not significantly different from G4 (CGMP-EAA-LP) (p = 0.45). Intake of water in G5 (FSAA-LP) was significantly higher than in G4 (CGMP-EAA-LP) and G3 (N), when compared separately (p<0.01). Mean values (week1-11) of food and water intake pr. mouse/week (Mean ± SD) is shown in Fig 2A and 2B. There was a strong positive correlation between mean values of intake of food and water.

Fig 2 — A: Average intake (g) of food pr. mouse/gr./w (w 1–11), Mean ± SD. G1 (13.6 +/- 3.1), G2 (15.4 +/- 2.5), G3 (13 (+/- 2.5), G2>G3 (p = 0.01), G4 (14.6 +/- 2.9), and G5 (15.4 +/- 2.8), G5>G3 (p = 0.01)). B: Average intake (g) of water pr. mouse/gr./w (w 1–11), Mean ± SD. G1 (29.5 (+/- 8.3); G2 (50.2 (+/- 20.3), G2>G1 (p< 0.001); G3 28.1 (+/- 6.9), G2>G3 (p< 0.001); G4 (30.3 (+/- 4.2), G5 (38.2 (+/- 5.7), G5>G3, G5>G4 (p≤0.001). C: Body Weight data (BW) (g) (weight start, weight end and weight gain) pr. mouse/gr./w (w1-12), Mean ± SD. Weight start: G1 (8.3 +/- 2.4), G2 (8.3 +/- 1.3), G3 (7.2 +/- 1.4), G4 (6.7 +/- 2.1) and G5 (7.4 +/- 2.5); Weight end: G1 (13.6 +/- 1.5), G2 (15.2 +/- 1.1), G3 (12.7 +/- 2.1), G4 (16.1 +/- 1.6) and G5 (16.6 +/- 0.9); Weight gain: G1 (5.4 +/- 2.5), G2 (6.9 +/- 1.3), G3 (5.4 +/- 2.3), G4 (9.3 +/- 1.9) and G5 (9.2 +/- 1.9). G2> G1 (p<0.05), G2> G3 (N) (p<0.01), G4 and G5 both > G3 (p ≤ 0.001). D: Body composition data, Body scan (g) (fat, lean body weight (LBW), weight total (WT)) pr. mouse/gr./w (w1-12) Mean ±SD. Fat: G1 (1.5 +/- 0.4), G2 (1.8 +/- 0.3), G3 (1.5 +/- 0.4), G4 (2.1 +/- 0.5), G5 (2.2 +/- 0.4), G4>G3 (p = 0.02), G5>G3 (p < 0.01)); LBW: G1 (12 +/- 1.3), G2 (13 +/- 1), G3 (10.8 +/- 1.5), G4 (13.5 +/- 0.9), G5 (13.7 +/- 0.7), G2>G3 (p < 0,01), G4>G3 (p < 0.001), G5>G3 (p < 0.0001)); WT: G1 (14 +/- 1.7), G2 (15.3 +/- 1.2), G3 (12.9 +/- 1.9), G4 (16.3 +/- 1.4), G5 (16.7 +/- 1), G2>G3 (p < 0,01), G4>G3 (p < 0.001), G5>G3 (p < 0.0001). E-G: DEXA scanning of the right femora, presented as (E) length (mm), (F) The bone mineral content (BMC) (g), and (G) areal bone mineral density (aBMD) (g/cm2) of the right femora. Mean ± SD. E (length, mm): G1 (13.52 +/- 0.28), G2 (13.82 +/- 0.39), G3 (13.15 +/- 0.77), G4 (14.21 +/- 0.30) and G5 (14.34 +/- 0.04), G4>G3 (p < 0.01)), G5>G3 (p <0.05). FBMC (g): G1 (0.0126 +/- 0.0009), G2 (0.0133 +/- 0.0019), G3 (0.0116 +/- 0.0023), G4 (0.0152 +/- 0.0021) and G5 (0.0160 +/- 0.0009), G4>G3 (p = 0.01), G5>G3 (p < 0.05). G aBMD (g/cm2): G1 (0.0463 +/- 0.0016), G2 (0.0481 +/- 0.0021), G3 (0.0452 +/- 0.0026), G4 (0.0503 +/- 0.0029) and G5 (0.0511 +/- 0.0024), G2>G3 (p <0.05), G4>G3 (p = 0.001), G5>G3(p<0.001). G1-G5 represent G1(N-CGMP), G2 (N-CGMP-LNAA), G3 (N), G4 (CGMP-EAA-LP) and G5 (FSAA-LP).

Growth (weight start, weight end, weight gain) and body scan (fat, lean and weight)

As expected, due to randomization of the litter into the five groups, there was no significant difference between the start weight for the five groups. At the end of the study, G2 (N-CGMP-LNAA) had a significantly higher weight compared to G1 (N-CGMP) (p<0.05) and G3 (N) (p<0.01). G4 (CGMP-EAA-LP) and G5 (FSAA-LP) were significantly higher for both weight end and weight gain compared to G3(N) (p ≤ 0.001). G4 (CGMP-EAA-LP) demonstrated the highest average growth compared to start weight (160.4%), followed by G5 (FSAA-LP) (146.5%) (Fig 2C). The same pattern was obtained for body composition of Lean (Fig 2D). G4 (CGMP-EAA-LP) and G5 (FSAA-LP) demonstrated the highest growth, indicating that CGMP and FSAA in combination with LP diet provide the best thrive. Our findings support the study by Ney et al, 2008 [12] showing a similar growth on CGMP compared to FSAA [12, 32].

Bone density measurements/bone examination

Body scan supported the thrive results. Femur length (mm), (BMC) (g) and aBMD (g/cm²) were significantly higher for G4 (CGMP-EAA-LP) and G5 (FSAA-LP) compared to G3(N). Furthermore, G2 (N-CGMP-LNAA) was significantly higher compared to G3 (N) for aBMD.

G4 (CGMP-EAA-LP) and G5 (FSAA-LP) revealed the highest value for BMC, aBMD and length of femora, which potentially indicate, that it is caused by the LP diet and supplement from either CGMP or FSAA, (p > 0.05 for all comparisons). All results are presented in Fig 2E–2G. Solverson et al documented similar increased bone strength in PKU mice after treatment with cGMP [32].

Weekly Phe and Tyr plasma analyses (Dried Blood Spot (DBS)

The Phe concentration was lower in group G1 (N-CGMP) and G2 (N-CGMP-LNAA) compared to G3 (N) at all timepoints (week 1–11), but only at week 2, G3 (N) had significantly higher levels of plasma Phe compared to G1(N-CGMP). There was no significant difference in Phe concentrations between G3 (N) and G2 (N-CGMP-LNAA). By comparison of G1(N-CGMP) with G2 (N-CGMP-LNAA) no significant difference in Phe concentration was observed, except at week 9, where G1 (N-CGMP) had significantly higher plasma Phe level compared to G2 (N-CGMP-LNAA).

At all timepoints, the Phe concentration in G4 (CGMP-EAA-LP) and G5 (FSAA-LP), were significantly lower compared to G3 (N). Significantly higher Phe concentration was observed in G5 (FSAA-LP) compared to G4 (CGMP-EAA-LP) at all time points, except for week 2, 4 and 11; Fig 3A

The concentrations of Tyr were similar for all groups, Fig 3B.

Due to lack of conversion of Phe to Tyr, affected individuals with PKU have an elevated Phe/Tyr ratio (typically >2.0 when untreated). The Phe/Tyr-ratio was significantly higher in G3 (N) compared to G4 (CGMP-EAA-LP), and G5 (FSAA-LP), Fig 3C.

Total AA profile (week 6 and 12 of the diet intervention period)

It was not possible to take a blood test for determination of the total amino acid profile at week 1 of diet intervention since the mice at that time, were too small to tolerate blood sampling, but as all animals were treated equal before week 1, we assume that the start value was similar in all groups.

Total amino acid profile was obtained for all animals at week 6 and week 12 (Fig 4), including the LNAAs of special interest: Tyr, Trp, Leu (added extra to G2 (N-CGMP-LNAA), G4 (CGMP-EAA-LP) and G5 (FSAA-LP)), Ile, Thr, Val (CGMP contains high amounts of these AAs), Arg, His, Met, Lys (added extra in G4 (CGMP-EAA-LP) and G5 (FSAA-LP)) and Phe.

Comparison between groups at week 6 and at week 12, respectively, for the same AA revealed the following results: No significant differences between G1 (N-CGMP) and G2 (N-CGMP-LNAA) neither at week 6 or week 12 were observed, but compared to G3 (N), there were quite a few. Phe was significant lower in G1 and G2 (N-CGMP-LNAA) compared to G3 (N) at week 12, whereas Phe was significantly lower in G2 (N-CGMP-LNAA) (but not in G1 ((N-CGMP)) compared to G3 (N) at week 6. Furthermore, Thr was significantly higher in both G1 (N-CGMP) and G2 (N-CGMP-LNAA) at week 6 compared to G3 (N), whereas Trp was significantly higher in both G1 (N-CGMP) and G2 (N-CGMP-LNAA) compared to G3(N) at week 12. In addition, Thr and Leu was also significantly higher in G2 (N-CGMP-LNAA), but not in G1 (N-CGMP) compared to G3 (N) at week 12.

It is possible that the increased amount of Lys in G2 (N-CGMP-LNAA), compared to G3 (N), although only significant level was obtained at week 12, might contribute to the significantly lower Phe level in G2 (N-CGMP-LNAA) already at week 6 compared to G3 (N).

By comparison of G3 (N), G4 (CGMP-EAA-LP) and G5 (FSAA-LP), we found that Phe was significantly higher in G3 (N) compared to G4 (CGMP-EAA-LP) and G5 (FSAA-LP) both at week 6 and at week 12, and Phe was significantly higher in G5 (FSAA-LP) compared to G4 (CGMP-EAA-LP) at week 12. The AA Ile and Leu was significantly higher in G4 (CGMP-EAA-LP) compared to G5 (FSAA-LP) both at week 6 and at week 12. These might contribute to the higher plasma concentration in G5 (FSAA-LP). P-values for comparison of subgroups are presented in Table 3

Table 3. P-values for comparison of the different groups G1-G5, representing G1(N-CGMP), G2 (N-CGMP-LNAA), G3 (N), G4 (CGMP-EAA-LP) and G5 (FSAA-LP).

Week 6	G1 & G2	G1 & G3		G2 & G3		G4 & G5		G4 & G3		G5 & G3
Lys	ns	ns		ns		ns		ns		ns
Arg	ns	ns		ns		ns		p < 0.05	G4>G3	ns
His	ns	ns		ns		ns		ns		p < 0.05	G5<G3
Trp	ns	ns		ns		ns		p < 0.01	G4>G3	p < 0.01	G5>G3
Thr	ns	p < 0.05	G1>G3	p < 0.05	G2>G3	ns		p < 0.01	G4>G3	p < 0.01	G5>G3
Val	ns	ns		ns		ns		ns		p < 0.01	G5<G3
Met	ns	ns		ns		ns		ns		ns
Ile	ns	ns		ns		p < 0.05	G4>G5	ns		p < 0.01
Leu	ns	ns		ns		p < 0.05	G4>G5	ns		ns
Tyr	ns	ns		ns		ns		ns		ns
Phe	ns	ns		p < 0.01	G3>G2	p = 0.05		p < 0.01	G4<G3	p < 0.01	G5<G3
Week 12	G1 & G2	G1 & G3		G2 & G3		G4 & 5		G4 & G3		G5 & G3
Lys	ns	ns		p < 0.01	G2>G3	ns		p < 0.05		p < 0.01	G5>G3
Arg	ns	ns		p < 0.05	G2>G3	ns		p < 0.01	G4>G3	p < 0.01	G5>G3
His	ns	ns		ns		ns		ns		ns
Trp	ns	p < 0.01	G1>G3	p < 0.01	G2>G3	ns		p < 0.01	G4>G3	p < 0.01	G5>G3
Thr	ns	ns		p < 0.05	G2>G3	ns		p < 0.01	G4>G3	p < 0.01	G5>G3
Val	ns	ns		ns		p < 0.01	G4>G5	p < 0.01	G4>G3	ns
Met	ns	ns		ns		ns		p < 0.05	G4>G3	ns
Ile	ns	ns		ns		p < 0.01	G4>G5	p < 0.01	G4>G3	ns
Leu	ns	ns		p < 0.05	G2>G3	p < 0.01	G4>G5	p < 0.01	G4>G3	ns
Tyr	ns	ns		ns		ns		ns		ns
Phe	ns	p < 0.01	G1<G3	p < 0.01	G2<G3	p < 0.01	G4<G5	p < 0.01	G4<G3	p < 0.01	G5<G3

Open in a new tab

Significant results are highlighted.

Also, the change from week 6 to week 12 were investigated for the different subgroups. By comparing changes for the individual AA’s between week 6 and 12, Trp increased significantly in G1(N-CGMP) and G2(N-CGMP-LNAA) from week 6 to week 12, and Leu increased significantly in G2(N-CGMP-LNAA).

Most significant changes were found for G4 (CGMP-EAA-LP) and G5 (FSAA-LP). Ile and Val increased significantly in both groups from week 6 to week 12 which could potentially be due to the high content of these AA in CGMP in G4 and as FSAA in G5, but also the majority of the extra added AAs (Leu, Met, His and Arg) in G4 (CGMP-EAA-LP) and G5 (FSAA-LP) increased significantly (p<0.05).

As expected, Phe increased significantly for G3 (N), but surprisingly also for G5 (FSAA-LP), which theoretically could be related to the slightly higher intake of Phe-containing water; 3.11 mg/day for G5 (FSAA-LP) compared to 2.47 mg/day for G4 (CGMP-EAA-LP). However, this did not prevent the other LNAA from increasing also. Mean intake of food varied from 13.0 (G3) to 15.4 (G5) g/week. Concentrations of selected AA in plasma presented as mean with SD for week 6 (sample 1) and week 12 (sample 2), difference between sample 1 and 2 and %-change and p-values for comparison of week 6 and week 12 are presented in Table 4.

Table 4. Plasma content of selected AA in plasma measured at week 6 and 12 presented as mean and SD, difference from start to end and % change.

ID (group)	Week 6 (μM/l)	Week 12 (μM/l)	difference	% change	p-value
	Phe
1	2665 +/- 270	2533 +/- 214	-132	-5	ns
2	2444 +/- 200	2296 +/- 289	-149	-6	ns
3	2828 +/- 253	3253 +/- 381	425	15	p = 0.01
4	580 +/- 243	601 +/- 189	21	4	ns
5	800 +/- 167	1009 +/- 126	209	26	p = 0.01
	Tyr
1	49 +/- 10	49 +/- 15	0	0	ns
2	48 +/- 3	48 +/- 7	0	0	ns
3	62 +/- 33	55 +/- 16	-7	-11	ns
4	48 +/- 11	64 +/- 26	17	35	ns
5	53 +/- 11	59 +/- 14	6	11	ns
	Trp
1	52 +/- 10	61 +/- 4	9	17	p<0.05
2	56 +/- 8	64 +/- 5	8	15	p<0.05
3	50 +/- 8	43 +/- 8	-7	-13	ns
4	73 +/- 20	87 +/- 9	14	19	ns
5	81 +/- 10	88 +/- 8	7	9	ns
	Leu
1	96 +/- 22	101 +/- 19	5	5	ns
2	88 +/- 17	117 +/- 16	29	33	p<0.01
3	93 +/- 8	96 +/- 14	3	3	ns
4	109 +/- 24	138 +/- 17	29	26	p = 0.01
5	88 +/- 6	103 +/- 15	15	17	p<0.05
	Met
1	46 +/- 8	49 +/- 10	3	6	ns
2	47 +/- 5	52 +/- 8	5	12	ns
3	50 +/- 8	48 +/- 7	-2	-4	ns
4	46 +/- 6	58 +/- 9	12	27	p<0.01
5	45 +/- 4	53 +/- 3	8	17	p<0.01
	Arg
1	59 +/- 25	72 +/- 35	14	23	ns
2	72 +/- 12	85 +/- 20	13	18	ns
3	64 +/- 32	59 +/- 23	-6	-9	ns
4	92 +/- 8	107 +/- 9	15	16	p<0.01
5	87 +/- 10	106 +/- 16	18	21	p = 0.01
	Lys
1	305 +/- 51	306 +/- 77	1	0	ns
2	319 +/- 38	344 +/- 47	25	8	ns
3	298 +/- 60	275 +/- 36	-23	-8	ns
4	293 +/- 35	318 +/- 31	24	8	ns
5	309 +/- 50	351 +/- 47	41	13	ns
	His
1	66 +/- 8	71 +/- 11	4	7	ns
2	64 +/- 7	75 +/- 7	11	18	p<0.01
3	71 +/- 13	71 +/- 7	0	0	ns
4	60 +/- 15	74 +/- 10	15	25	p<0.05
5	58 +/- 6	70 +/- 8	12	21	p<0.01
	Thr
1	210 +/- 46	209 +/- 57	-1	0	ns
2	215 +/- 42	204 +/- 38	-10	-5	ns
3	167 +/- 33	169 +/- 26	2	1	ns
4	326 +/- 84	458 +/- 240	132	40	ns
5	367 +/- 175	452 +/- 98	84	23	ns
	Val
1	177 +/- 32	167 +/- 26	-10	-6	ns
2	169 +/- 17	191 +/- 25	22	13	ns
3	173 +/- 14	168 +/- 22	-5	-3	ns
4	168 +/- 38	216 +/- 31	48	29	p = 0.01
5	148 +/- 11	166 +/- 14	18	12	p = 0.01
	Ile
1	79 +/- 15	80 +/- 10	0	0	ns
2	73 +/- 10	95 +/- 24	22	30	p<0.05
3	78 +/- 9	74 +/- 13	-4	-5	ns
4	76 +/- 14	101 +/- 17	25	33	p<0.01
5	62 +/- 6	73 +/- 10	12	19	p<0.05

Open in a new tab

Group 1–5 represent G1(N-CGMP), G2 (N-CGMP-LNAA), G3 (N), G4 (CGMP-EAA-LP) and G5 (FSAA-LP).

Brain examination

The following brain parts were examined: Cerebellum, brain stem, hypothalamus, parietal cortex, anterior piriform, cortex and olfactory bulb. We tested for the following AA and their metabolites: Phe, Tyr, Trp, 5-HT, 5-HIAA, DA and DOPAC. The amino acids, Trp, Tyr and Phe utilize the same LAT1 transporter over the BBB.

Trp is metabolized to 5-HT in the brain and finally further metabolized mainly to 5-HIAA. Tyr is precursor of DA which is further converted to DOPAC. These metabolites are crucial in order to evaluate the LNAA balance in the brain.

The content of DA, 5-HT, 5-HIAA, DOPAC, Phe, Tyr and Trp (Mean ± SD) for total brain, by ading values for the individual brain parts together is presented in Fig 5.

Fig 5 — Trp is metabolized to 5-HT in the brain and finally further metabolized mainly to 5-HIAA. Tyr is precursor of DA which is further converted to DOPAC. Group 1–5 represent: G1(N-CGMP), G2 (N-CGMP-LNAA), G3 (N), G4 (CGMP-EAA-LP) and G5 (FSAA-LP).

Overall, G3 (N) had the highest Phe levels and G4 (CGMP-EAA-LP) had the lowest in the entire brain followed by G5 (FSAA-LP). The concentration of Phe in G3 (N) was significantly increased compared to all the other groups.

In contrast, Tyr in total brain levels only showed minor variations between the groups despite the different content in the five chow (0.67–1.44 g AA/100 g).

Trp levels were significantly higher in G1 (N-CGMP) compared to G2 (N-CGMP-LNAA), and significantly higher in G5 (FSAA-LP) compared to G3 (N) and G4 (CGMP-EAA-LP). DOPAC only reached significant increased level in G5 (FSAA-LP) compared to G3 (N) (p < 0.05). DA did not reach significant level at all. 5-HT and 5-HIAA both demonstrated a 2-3-fold higher value in G4 and G5 (FSAA-LP) compared to G1(N-CGMP), G2 (N-CGMP-LNAA), and G3 (N).

Investigation of the individual brain parts revealed a significant effect compared to control G3(N) for all groups on most brain parts for Phe. G1 (N-CGMP) and G2(N-CGMP-LNAA) had almost similar levels in all brain parts, and no significant differences were found (Fig 6).

Fig 6 — Trp is metabolized to 5-HT in the brain and finally further metabolized mainly to 5-HIAA. Tyr is precursor of DA which is further converted to DOPAC.

The concentration of Tyr and Trp (34) in the brain theoretically determine the brain concentration of DA and 5-HT respectively. Both Trp and Tyr levels were significantly increased in the brain stem (BS) in G1(N-CGMP) compared to G2(N-CGMP-LNAA). In spite of this, 5HT was significantly increased in several brain parts (anterior piriform (AP), brain stem (BS), hypothalamus (HT) in G2 (N-CGMP-LNAA) compared to G1 (N-CGMP). Also, significantly higher levels of 5-HT in G2 (N-CGMP-LNAA) compared to G3(N) were observed in most brain parts. The concentration of 5-HIAA was increased significantly in several brain parts in both G1(N-CGMP) and G2 (N-CGMP-LNAA) compared to G3(N). P-values are presented in Table 5. Thus, no clear correlation between the Trp, 5HT and 5-HIAA concentration exist in G1(N-CGMP) and G2 (N-CGMP-LNAA). In contrast, the higher concentration of Trp in G4 (CGMP-EAA-LP) and G5 (FSAA-LP) compared to G3 (N) also leads to higher 5-HT concentration in G4 (CGMP-EAA-LP) and G5 (FSAA-LP). Also, the concentration of 5HIAA was higher in the brain of G4 (CGMP-EAA-LP) and G5 (FSAA-LP), compared to G3 (N).

Table 5. Difference in concentrations of selected AA and metabolites in different brain parts.

	Anterior piriform			Brain stem			Bulbus olfactorius			Cerebellum			Hypothalamus			Parietal cortex
	compound	p value		compound	p value		compound	p value		compound	p value		compound	p value		compound	p value
1,2	Phe	0.7879		Phe	0.7998		Phe	0.8945		Phe	0.8953		Phe	0.2752		Phe	0.5250
1,2	Tyr	0.7564		Tyr	0.0085	1 > 2	Tyr	0.6638		Tyr	0.0970		Tyr	0.5201		Tyr	0.1666
1,2	Trp	0.6513		Trp	0.0116	1 > 2	Trp	0.5298		Trp	0.2778		Trp	0.3170		Trp	0.2335
1,2	DA	0.1887		DA	0.2201		DA	0.2061		DA	0.6869		DA	0.0563		DA	0.5618
1,2	.5-HT	0.0012	2 > 1	.5-HT	0.0044	2 > 1	.5-HT	0.0297		.5-HT	0.7493		.5-HT	0.0345	2 > 1	.5-HT	0.6193
1,2	5-HIAA	0.8853		5-HIAA	0.7697		5-HIAA	0.0641		5-HIAA	0,0311	2 > 1	5-HIAA	0.3304		5-HIAA	0.2547
1,2	DOPAC	0.8451		DOPAC	0.8805		DOPAC	0.1396		DOPAC	0.2795		DOPAC	0.9644		DOPAC	0.1718
1,3	Phe	0.0133	3 > 1	Phe	0.0004	3 > 1	Phe	0.0186	3 > 1	Phe	0.0025	3 > 1	Phe	0.0845		Phe	0.0043	3 > 1
1,3	Tyr	0.8724		Tyr	0.1502		Tyr	0.3975		Tyr	0.8367		Tyr	0.9032		Tyr	0.4341
1,3	Trp	0.7648		Trp	0.4980		Trp	0.7689		Trp	0.8210		Trp	0.7913		Trp	0.5858
1,3	DA	0.0711		DA	0.2912		DA	0.2695		DA	0.9945		DA	0,7134		DA	0.3549
1,3	.5-HT	0,8222		.5-HT	0.2320		.5-HT	0.8345		.5-HT	0.8833		.5-HT	0.5810		.5-HT	0.0290	1 > 3
1,3	5-HIAA	0.0695		5-HIAA	0.9944		5-HIAA	0.2008		5-HIAA	0,0115	1 > 3	5-HIAA	0.0346		5-HIAA	0,0114	1 > 3
1,3	DOPAC	0.7140		DOPAC	0.9410		DOPAC	0.3591		DOPAC	0.6047		DOPAC	0.9589		DOPAC	0.4987
2,3	Phe	0.0408	3 > 2	Phe	0.0006	3 > 2	Phe	0.0560		Phe	0.0003	3 > 2	Phe	0.0056	3 > 2	Phe	0.0041	3 > 2
2,3	Tyr	0.9308		Tyr	0.0037	3 > 2	Tyr	0.6715		Tyr	0.0990		Tyr	0.5060		Tyr	0.5123
2,3	Trp	0.9542		Trp	0.2269		Trp	0.4101		Trp	0.4390		Trp	0.6717		Trp	0.8999
2,3	DA	0.8181		DA	0.6245		DA	0.8394		DA	0.6265		DA	0.0248	2 > 3	DA	0.8466
2,3	.5-HT	0.0002	2 > 3	.5-HT	0.0814		.5-HT	0.0371	2 > 3	.5-HT	0.5343		.5-HT	0.0024	2 > 3	.5-HT	0.0012	2 > 3
2,3	5-HIAA	0.0018	2 > 3	5-HIAA	0.8356		5-HIAA	0.0153	2 > 3	5-HIAA	0.2328		5-HIAA	0.0356	2 > 3	5-HIAA	0.0018	2 > 3
2,3	DOPAC	0.6253		DOPAC	0.8220		DOPAC	0.4868		DOPAC	0.4190		DOPAC	0.9860		DOPAC	0.3472
3,4	Phe	0.0000	3 > 4	Phe	0.0000	3 > 4	Phe	0.0000	3 > 4	Phe	0.0000	3 > 4	Phe	0.0000	3 > 4	Phe	0.0000	3 > 4
3,4	Tyr	0.8089		Tyr	0.0025	4 > 3	Tyr	0.8022		Tyr	0.1360		Tyr	0.3538		Tyr	0.9469
3,4	Trp	0.1889		Trp	0.8038		Trp	0.3170		Trp	0.9670		Trp	0.1782		Trp	0.1453
3,4	DA	0.1572		DA	0.6240		DA	0.8389		DA	0.0301	4 > 3	DA	0.1563		DA	0.0046	4 > 3
3,4	.5-HT	0.0000	4 > 3	.5-HT	0.0024	4 > 3	.5-HT	0.0003	4 > 3	.5-HT	0.0021	4 > 3	.5-HT	0.0001	4 > 3	.5-HT	0.0000	4 > 3
3,4	5-HIAA	0.0000	4 > 3	5-HIAA	0.0001	4 > 3	5-HIAA	0.0001	4 > 3	5-HIAA	0.0014	4 > 3	5-HIAA	0.0000	4 > 3	5-HIAA	0.0000	4 > 3
3,4	DOPAC	0.4708		DOPAC	0.8229		DOPAC	0.8374		DOPAC	0.2559		DOPAC	0.7765		DOPAC	0.0648
3,5	Phe	0.0000	3 > 5	Phe	0.0000	3 > 5	Phe	0.0000	3 > 5	Phe	0.0000	3 > 5	Phe	0.0000	3 > 5	Phe	0.0000	3 > 5
3,5	Tyr	0.0061	5 > 3	Tyr	0.2414		Tyr	0.3133		Tyr	0.4329		Tyr	0.2761		Tyr	0.0132	5 > 3
3,5	Trp	0.0127	5 > 3	Trp	0.0978		Trp	0.2009		Trp	0.4123		Trp	0.0421		Trp	0.0331	5 > 3
3,5	DA	0.9627		DA	0.9576		DA	0.5314		DA	0.1247		DA	0.0110	5 > 3	DA	0.0011	5 > 3
3,5	.5-HT	0.0001	5 > 3	.5-HT	0.1094		.5-HT	0.0022	5 > 3	.5-HT	0.0010	5 > 3	.5-HT	0.0003	5 > 3	.5-HT	0.0000	5 > 3
3,5	5-HIAA	0.0000	5 > 3	5-HIAA	0.0019	5 > 3	5-HIAA	0.0000	5 > 3	5-HIAA	0.0006	5 > 3	5-HIAA	0.0000	5 > 3	5-HIAA	0.0000	5 > 3
3,5	DOPAC	0.1917		DOPAC	0.1836		DOPAC	0.1790		DOPAC	0.0044	5 > 3	DOPAC	0.1430		DOPAC	0.0501	5 > 3
4,5	Phe	0.1217		Phe	0.2863		Phe	0.8234		Phe	0.3462		Phe	0.6233		Phe	0.2721
4,5	Tyr	0.0019	5 > 4	Tyr	0.0090	5 > 4	Tyr	0.2326		Tyr	0.0146	5 > 4	Tyr	0.0341	5 > 4	Tyr	0.0135	5 > 4
4,5	Trp	0.0439	5 > 4	Trp	0.0320	5 > 4	Trp	0.6502		Trp	0.2831		Trp	0.2502		Trp	0.2713
4,5	DA	0.1525		DA	0.6086		DA	0.6899		DA	0.5418		DA	0.6666		DA	0.8516
4,5	.5-HT	0.1272		.5-HT	0.1041		.5-HT	0.0489	4 > 5	.5-HT	0.3136		5-HT	0.3208		.5-HT	0.0870
4,5	5-HIAA	0.1394		5-HIAA	0.1556		5-HIAA	0.0277	4 > 5	5-HIAA	0.2488		5-HIAA	0.5349		5-HIAA	0.6324
4,5	DOPAC	0.5633		DOPAC	0.1696		DOPAC	0.2185		DOPAC	0.0378	5 > 4	DOPAC	0.2968		DOPAC	0.5607

Open in a new tab

Significant results for comparisons between groups for Phe (μg/g), Tyr (μg/g), Trp (μg/g), 5-HIAA(ng/g), DA (ng/g), DOPAC(ng/g) and 5-HT(ng/g) in all the individual brain parts (anterior piriform (AP), brain stem (BS), olfactory bulb (BO), Cerebellum (CE), hypothalamus (HT) & parietal cortex (PC). The numbers 1–5 represent G1(N-CGMP), G2 (N-CGMP-LNAA), G3 (N), G4 (CGMP-EAA-LP) and G5 (FSAA-LP).

Although the concentration of Tyr was significantly increased in several brain parts in G5 (FSAA-LP) compared to G4 (CGMP-EAA-LP) this was not associated with an increase in neither the concentration of DA nor DOPAC (Fig 6).

Comparison of the total brain concentration and plasma concentration of Phe indicated a strong correlation between the concentration in the blood and the brain (Fig 7A). Also, the concentration of Trp in plasma and brain indicate correlation (Fig 7C). A high concentration of Trp in the blood were associated with a high concentration in the brain. For Tyr there was no clear correlation (Fig 7C).

The plasma Phe values were in line with the concentration-levels found in the total brain, indicating, that the concentration in plasma is a major factor determining the concentration of Phe in the brain while the relation between plasma- and brain-concentration of Tyr and Trp, respectively, is determined by other factors, as previously documented by Berry, Harding and Pacussi [33–35].

No clear correlation could be observed when investigation of the brain concentration of Trp with the brain concentration of 5-HT (Fig 7D). 5-HIAA is the main metabolite of 5-HT [36] and we did find a strong correlation (0.87) (Fig 7E). Despite this, the correlation between the concentration of 5-HT and 5-HIAA in G4 (CGMP-EAA-LP) and G5 (FSAA-LP) was not obvious (Fig 7E). Investigation of the brain concentration of Tyr and DA and of DA and DOPAC revealed no correlation, as expected (Fig 7F and 7G).

The lack of clear correlations between the concentration of Trp and Tyr and its metabolites, are in agreement with observations published by Harding et al., argued that the most likely cause of brain DA and 5-HT deficiency in PKU is Phe-mediated inhibition of brain Tyr hydroxylase (TH) and Trp hydroxylase (TPH) activities rather than decreased substrate availability [35]. The lack of any significant difference according to the concentration of DA, is consistent with the findings of Puglisi-Allegra et al., demonstrating that DA is the least affected brain amine in PKU mice [37].

Behavioral test (Barnes maze)

Metrics measured for the behavioral studies were: 1) Duration of each trial (Fig 8A) 2) the mean speed of the animal (Fig 8B) and 3) the distance travelled (Fig 8C). Unlike test duration, the mean speed of the animals did not seem to alter during testing. See legend, Fig 8 for significant differences.

Exclusion of animals with malocclusion due to incisor overgrowth

A high incidence of malocclusion due to overgrowth of the incisors was found in this type of PKU mice. The regular contact due to body weight measurement made it possible to diagnose the presence of these lesions, and a programme of regular tooth trimming using blunt-tipped scissors was used to cut the individuals with long incisors. It occurred in all groups and varied from 21–40 times per group (p = 0.38), or 0–12 times per mouse during the 12-week test period. Total intake of food was not related to number of teeth cuts. The highest number of cuts occurred in G4 (CGMP-EAA-LP) (40 times), followed by G2 (N-CGMP-LNAA), (27 times), G3 (N) (26 times), G1(N-CGMP), (21 times) and G5 (FSAA-LP), (14 times) with the lowest number of cuts.

Discussion

Performing study 1, the first question we wanted to clarify was if pure CGMP or CGMP supplemented with the LNAAs given off diet are able to lower the content of Phe in the brain. As both G1 (N-CGMP) and G2 (N-CGMP-LNAA), had significantly lower plasma and brain Phe concentration compared to G3 (N), at week 12. This study confirmed that mice off diet supplemented with CGMP had significantly lower Phe in plasma and total brain compared to a control group fed a normal diet. This is important, since many PKU patients worldwide are off diet, but could benefit from CGMP treatment.

Other studies conducted in wild type and PKU mice fed diets containing either 20% protein from casein, Phe-free synthetic AA or CGMP supplemented with limiting indispensable AA have revealed promising results. In one study, concentrations of Phe in the plasma decreased by 11% and in 5 regions of the brain by 20% when compared to Phe-free synthetic AA [12].

The second question we wanted to clarify was if the supplementation of extra LNAA results in significantly lower brain Phe level compared to supplementation of CGMP alone. At week 6 the Phe concentration was significantly lower in G2 (N-CGMP-LNAA) (but not in G1 (N-CGMP)) compared to G3 (N), but the only significant difference between G1 (N-CGMP) and G2 (N-CGMP-LNAA) was obtained at week 9, where the plasma Phe concentration was significantly higher in G1 (N-CGMP) compared to G2 (N-CGMP-LNAA). Based on these results we might conclude that the additional LNAA did not have any big impact on the brain Phe concentration.

A large number of studies have confirmed, that LNAA have the ability to reduce Phe entering the brain [18, 19, 38–41]. As specified in the beginning, the Phe blocking effect between blood and brain were relevant to investigate for G1(N-CGMP) and G2 (N-CGMP-LNAA), since these diets had very similar composition, except that LNAA was added solely to G2 (N-CGMP-LNAA). The concentration of plasma Trp increased significantly in both G1 (N-CGMP) and G2 (N-CGMP-LNAA), from week 6 to week 12, and both groups showed similar levels of Tyr and Trp in both plasma and brain. No increase in concentration of plasma Trp was observed for G3 (N). Since CGMP already contain large amounts of Thr, Val and Ile, these AAs could potentially be sufficient to block passage of Phe across the BBB. However as the Phe concentration in det given to G3(N) contains more Phe compared to both G1 (N-CGMP) and G2(N-CGMP-LNAA), (1.01 versus 0.74 g/100g; approximately 36% more) this difference could also account for the increased plasma Phe concentration at week 12 (3253 versus 2533 and 2296 μM/l, approximately 28–41% more).

However, some benefit from adding LNAA to the diet was observed according to the growth, bone density and brain serotonin (5-HT). At the end of the study, G2 (N-CGMP-LNAA) had a significantly higher weight compared to both G1 (N-CGMP) and G3 (N) and furthermore a significant increased aBMD was observed for G2 (N-CGMP-LNAA) compared to the control group, G3 (N).

The aim according to study 2 was to address the following question: Does a combination of CGMP, EAAs and LP diet, provide similar plasma and brain Phe levels, growth and behavioral skills as a formula with a similar combination of pure FSAA.

Both the plasma and the brain concentration of Phe in G4 CGMP-EAA-LP) and G5 (FSAA-LP) were significantly reduced compared to G3 (N). Furthermore, the diet containing CGMP, EAAs and LP diet seems more potent in reducing the plasma Phe concentration compared to the formula containing FSAA as the concentration of Phe in the plasma in G4 CGMP-EAA-LP) was significantly reduced compared to the concentration in G5 (FSAA-LP). However, this effect could be related to the higher intake of phe-containing water (26% more in G5(FSAA-LP), compared to G4 (CGMP-EAA-LP).

Investigation of the growth revaled that both formula leads to higher weight, BMC, aBMC and femur lengt compared to normal diet (N). Again the formula G4 (CGMP-EAA-LP) seems slighly more potent as the highest average growth compared to start was obtaind for G4 (CGMP-EAA-LP), compared to G5 (FSAA-LP).

Barnes Maze test has been successfully used in other mice studies [42, 43]. However, this test may not have been sensitive enough for this specific mouse strain, since it was (with a few exceptions) not possible to find any significant differences in behavior. The strain has been used in other studies at Aarhus University [44] and Bruinenberg et all found that the genetic background of the mice could play an important role [45]. An explanation could be, that the mice did not start the diet intervention before week 4, as diet intervention before this time is very difficult. A study has shown that week 3 is a very critical period and could potentially already have affected the brain [22]. Other explanations for this could be that the number of animals per group were too low and/or the test period too short to show a difference. Since the main objective of the study was to determine the differences between the diet compositions, we did not include a wild type mouse. However, this could have been useful for the maze study, since there were no differences between the groups, despite the different diet regimes. Similar studies have used wild type mouse to compare with [12, 46]

Thus al together we can conclude that a combination of CGMP, EAAs and LP diet, provide similar plasma and brain Phe levels, growth as a formula with a similar combination of pure FSAA. According to behavioral skills, we are unfortunately not able to make any conclusion.

It is noteworthy that several AA (Leu, Ile, Met, Val, His and Arg) increased significantly for both G4(CGMP-EAA-LP) and G5(FSAA-LP) from week 6 to week 12, and also that Ile, Leu and Val demonstrated significantly higher values for G4(CGMP-EAA-LP) compared to G5 (FSAA-LP) after week 12. This indicate that the higher level of Ile and Val reflect a better absorption due to the natural content of these specific AAs in CGMP.

The effect of the individual diets in G4(CGMP-EAA-LP) and G5 (FSAA-LP) on Phe levels are recognizable during the entire period, already after the first week of treatment. Since both G1(N-CGMP) and G2 (CGMP-LNAA-A) were off diet, we expected that Phe would be higher in these groups compared to G4 (CGMP-EAA-LP) and G5 (FSAA-LP).

The high Phe concentration in G3(N) was expected due to the casein diet. Phe increased significantly from week 6 to week 12 in G3 (N), which is in line with studies showing, that Phe builds up in plasma over time and results in irreversible brain damage. Our result demonstrate that the mice fed on normal diet (G3) have shorter femur length, lower BNC and lower aBMD indication that the high Phe level also might affect the bone formation negatively.

Conclusion

This study provides important data about CGMP supplementation as a possible alternative treatment strategy for PKU. This study verified that it was possible to lower brain Phe significantly with supplementation of CGMP to a semi free (normal) diet. Also, it was confirmed that CGMP in combination with FSAA as supplement to LP diet had the same effect on plasma- and brain Phe levels, growth as a formula with a similar combination of FSAA. Treatment of PKU is already successful, but new alternatives are necessary in order to improve compliance [47]. CGMP can provide a safe and efficient supplement to a semi free- as well as a LP diet as an alternative to FSAA products. Further long-term studies in humans are needed to support the findings from the present study.

Acknowledgments

The authors express their gratitude to Dorte Hermansen, Benedicte Vestergaard Jensen and Jani Kær for assistance in the animal facility.

Glossary

CGMP-20: (product name Lacprodan^® CGMP-20)
G1(N-CGMP): Normal (N) casein diet (75%) in combination with CGMP (25%)
G2 (N-CGMP-LNAA): Normal (N) casein diet (75%) in combination with CGMP (19,7%) and selected LNAA (5,3% Leu, Tyr and Trp)
G3 (N): normal casein diet (100%)
G4 (CGMP-EAA-LP): CGMP (70,4%) in combination with essential AA (19,6%) and LP diet
G5 (FSAA-LP): FSAA (100%) and LP diet

Data Availability

All relevant data are within the paper.

Funding Statement

The research was conducted as part of an lndustrial PhD program, which in Denmark is a three-year industrially focused PhD project where a student (Kirsten Ahring) is hired by a company (Arla Foods lngredients (AFI)) and enrolled at a university (Copenhagen University) at the same time. The industrial PhD program was managed by Innovation Fund Denmark and partly funded by AFI (https://ufm.dk/en/research-and-innovation/funding-proqrammesfor-research-and-innovation/find-danish-fundingprogrammes/programmes-managed-byinnovation-fund-denmark/industrial-phd). AFI produces and commercialize the food ingredient; Casein glycomacropeptide which was used in the research.

References

1.Blau N, Hennermann JB, Langenbeck U, Lichter-Konecki U. Diagnosis, classification, and genetics of phenylketonuria and tetrahydrobiopterin (BH4) deficiencies. Mol Genet Metab. 2011;104 Suppl:S2–9. [DOI] [PubMed] [Google Scholar]
2.Blau N, van Spronsen FJ, Levy HL. Phenylketonuria. Lancet. 2010;376(9750):1417–27. doi: 10.1016/S0140-6736(10)60961-0 [DOI] [PubMed] [Google Scholar]
3.Mitchell JJ, Trakadis YJ, Scriver CR. Phenylalanine hydroxylase deficiency. Genet Med. 2011;13(8):697–707. doi: 10.1097/GIM.0b013e3182141b48 [DOI] [PubMed] [Google Scholar]
4.Schuck PF, Malgarin F, Cararo JH, Cardoso F, Streck EL, Ferreira GC. Phenylketonuria Pathophysiology: on the Role of Metabolic Alterations. Aging Dis. 2015;6(5):390–9. doi: 10.14336/AD.2015.0827 [DOI] [PMC free article] [PubMed] [Google Scholar]
5.van Wegberg AMJ, MacDonald A, Ahring K, Bélanger-Quintana A, Blau N, Bosch AM, et al. The complete European guidelines on phenylketonuria: diagnosis and treatment. Orphanet journal of rare diseases. 2017;12(1):162-. doi: 10.1186/s13023-017-0685-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Demirdas S, Coakley KE, Bisschop PH, Hollak CEM, Bosch AM, Singh RH. Bone health in phenylketonuria: a systematic review and meta-analysis. Orphanet Journal of Rare Diseases. 2015;10(1):17. doi: 10.1186/s13023-015-0232-y [DOI] [PMC free article] [PubMed] [Google Scholar]
7.MacDonald A, Lilburn M, Davies P, Evans S, Daly A, Hall SK, et al. ’Ready to drink’ protein substitute is easier is for people with phenylketonuria. J Inherit Metab Dis. 2006;29(4):526–31. doi: 10.1007/s10545-006-0234-y [DOI] [PubMed] [Google Scholar]
8.Giovannini M, Verduci E, Salvatici E, Paci S, Riva E. Phenylketonuria: nutritional advances and challenges. Nutr Metab (Lond). 2012;9(1):7. doi: 10.1186/1743-7075-9-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
9.Zimmermann M, Jacobs P, Fingerhut R, Torresani T, Thöny B, Blau N, et al. Positive effect of a simplified diet on blood phenylalanine control in different phenylketonuria variants, characterized by newborn BH4 loading test and PAH analysis. Mol Genet Metab. 2012;106(3):264–8. doi: 10.1016/j.ymgme.2012.04.016 [DOI] [PubMed] [Google Scholar]
10.Aimutis WR. Bioactive properties of milk proteins with particular focus on anticariogenesis. J Nutr. 2004;134(4):989s–95s. doi: 10.1093/jn/134.4.989S [DOI] [PubMed] [Google Scholar]
11.Etzel MR. Manufacture and use of dairy protein fractions. The Journal of Nutrition. 2004;134(4):996S. doi: 10.1093/jn/134.4.996S [DOI] [PubMed] [Google Scholar]
12.Ney DM, Hull AK, van Calcar SC, Liu X, Etzel MR. Dietary glycomacropeptide supports growth and reduces the concentrations of phenylalanine in plasma and brain in a murine model of phenylketonuria. J Nutr. 2008;138(2):316–22. doi: 10.1093/jn/138.2.316 [DOI] [PubMed] [Google Scholar]
13.Van Calcar SC, Hull AK, Liu X, Etzel M, Ney DM. Dietary glycomacropeptide (GMP) supports growth and reduces the concentrations of phenylalanine in plasma and brain in the PKU mouse. Mol Genet Metab. 2008;93(3):263-. [DOI] [PubMed] [Google Scholar]
14.van Calcar SC, MacLeod EL, Gleason ST, Etzel MR, Clayton MK, Wolff JA, et al. Improved nutritional management of phenylketonuria by using a diet containing glycomacropeptide compared with amino acids. Am J Clin Nutr. 2009;89(4):1068–77. doi: 10.3945/ajcn.2008.27280 [DOI] [PMC free article] [PubMed] [Google Scholar]
15.van Calcar SC, Macleod EL, Gleason ST, Etzel MR, Rice GM, Ney DM. Glycomacropeptide (GMP): A new option for PKU diet management. Mol Genet Metab. 2011;102(3):262-. [Google Scholar]
16.van Calcar SC, Ney DM. Food Products Made with Glycomacropeptide, a Low-Phenylalanine Whey Protein, Provide a New Alternative to Amino Acid-Based Medical Foods for Nutrition Management of Phenylketonuria. J Acad Nutr Diet. 2012;112(8):1201–10. doi: 10.1016/j.jand.2012.05.004 [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Zaki OK, El-Wakeel L, Ebeid Y, Elarab HSE, Moustafa A, Abdulazim N, et al. The Use of Glycomacropeptide in Dietary Management of Phenylketonuria. Journal of Nutrition and Metabolism. 2016. doi: 10.1155/2016/2453027 [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Schindeler S, Ghosh-Jerath S, Thompson S, Rocca A, Joy P, Kemp A, et al. The effects of large neutral amino acid supplements in PKU: an MRS and neuropsychological study. Mol Genet Metab. 2007;91(1):48–54. doi: 10.1016/j.ymgme.2007.02.002 [DOI] [PubMed] [Google Scholar]
19.Rocha JC, Martel F. Large neutral amino acids supplementation in phenylketonuric patients. J Inherit Metab Dis. 2009;32(4):472–80. doi: 10.1007/s10545-009-1132-x [DOI] [PubMed] [Google Scholar]
20.Lou HC, Güttler F, Lykkelund C, Bruhn P, Niederwieser A. Decreased vigilance and neurotransmitter synthesis after discontinuation of dietary treatment for phenylketonuria in adolescents. Eur J Pediatr. 1985;144(1):17–20. doi: 10.1007/BF00491918 [DOI] [PubMed] [Google Scholar]
21.Lou HC, Lykkelund C, Gerdes AM, Udesen H, Bruhn P. Increased vigilance and dopamine synthesis by large doses of tyrosine or phenylalanine restriction in phenylketonuria. Acta Paediatr Scand. 1987;76(4):560–5. doi: 10.1111/j.1651-2227.1987.tb10521.x [DOI] [PubMed] [Google Scholar]
22.Pascucci T, Giacovazzo G, Andolina D, Accoto A, Fiori E, Ventura R, et al. Behavioral and neurochemical characterization of new mouse model of hyperphenylalaninemia. PLoS One. 2013;8(12):e84697. doi: 10.1371/journal.pone.0084697 [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Shedlovsky A, McDonald JD, Symula D, Dove WF. Mouse models of human phenylketonuria. Genetics. 1993;134(4):1205–10. doi: 10.1093/genetics/134.4.1205 [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Mekada K, Abe K, Murakami A, Nakamura S, Nakata H, Moriwaki K, et al. Genetic differences among C57BL/6 substrains. Exp Anim. 2009;58(2):141–9. doi: 10.1538/expanim.58.141 [DOI] [PubMed] [Google Scholar]
25.Ahring K, Kjolby M. Effect of offering Phenylketonuria (PKU) mice Glycomacropeptide (GMP) before normal or low protein (LP) diet on growth, serum-phenylalanine (phe)-concentration, bone structure, performance and concentration of phe in the brain. protocols.io doi: 10.17504/protocols.io.bkg9ktz6 [DOI] [Google Scholar]
26.Benevenga NJ, Gahl MJ, Crenshaw TD, Finke MD. Protein and amino acid requirements for maintenance and amino acid requirements for growth of laboratory rats. J Nutr. 1994;124(3):451–3. doi: 10.1093/jn/124.3.451 [DOI] [PubMed] [Google Scholar]
27.Johannik K, Vanhecke P, Francois B, Marchal G, Smet MH, Jaeken J, et al. Localized Brain Proton Nmr-Spectroscopy in Young-Adult Phenylketonuria Patients. Magn Reson Med. 1994;31(1):53–7. [DOI] [PubMed] [Google Scholar]
28.Protein and amino acid requirements in human nutrition. World Health Organ Tech Rep Ser. 2007(935):1–265, back cover. [PubMed] [Google Scholar]
29.Peake RW, Law T, Hoover PN, Gaewsky L, Shkreta A, Kellogg MD. Improved separation and analysis of plasma amino acids by modification of the MassTrak AAA Solution Ultraperformance(R) liquid chromatography method. Clin Chim Acta. 2013;423:75–82. doi: 10.1016/j.cca.2013.03.036 [DOI] [PubMed] [Google Scholar]
30.Pritchett K, Mulder GB. T, radial arm, and Barnes mazes. Contemp Top Lab Anim Sci. 2003;42(3):53–5. [PubMed] [Google Scholar]
31.Miller DJ. Sydney Ringer; physiological saline, calcium and the contraction of the heart. The Journal of Physiology. 2004;555(Pt 3):585–7. doi: 10.1113/jphysiol.2004.060731 [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Solverson P, Murali SG, Litscher SJ, Blank RD, Ney DM. Low bone strength is a manifestation of phenylketonuria in mice and is attenuated by a glycomacropeptide diet. PloS one. 2012;7(9):e45165–e. doi: 10.1371/journal.pone.0045165 [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Berry HK, Brunner RL, Hunt MM, White PP. Valine, isoleucine, and leucine. A new treatment for phenylketonuria. Am J Dis Child. 1990;144(5):539–43. doi: 10.1001/archpedi.1990.02150290033020 [DOI] [PubMed] [Google Scholar]
34.Pascucci T, Andolina D, Ventura R, Puglisi-Allegra S, Cabib S. Reduced availability of brain amines during critical phases of postnatal development in a genetic mouse model of cognitive delay. Brain Res. 2008;1217:232–8. doi: 10.1016/j.brainres.2008.04.006 [DOI] [PubMed] [Google Scholar]
35.Harding CO, Winn SR, Gibson KM, Arning E, Bottiglieri T, Grompe M. Pharmacologic inhibition of L-tyrosine degradation ameliorates cerebral dopamine deficiency in murine phenylketonuria (PKU). J Inherit Metab Dis. 2014;37(5):735–43. doi: 10.1007/s10545-013-9675-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Nakano T, Silva-Hernandez ER, Ikawa N, Ozimek L. Purification of kappa-casien glycomacropeptide from sweet whey with undetectable level of phenylalanine. Biotechnol Prog. 2002;18(2):409–12. doi: 10.1021/bp010195u [DOI] [PubMed] [Google Scholar]
37.Puglisi-Allegra S, Cabib S, Pascucci T, Ventura R, Cali F, Romano V. Dramatic brain aminergic deficit in a genetic mouse model of phenylketonuria. Neuroreport. 2000;11(6):1361–4. doi: 10.1097/00001756-200004270-00042 [DOI] [PubMed] [Google Scholar]
38.Matalon R, Michals-Matalon K, Bhatia G, Burlina AB, Burlina AP, Braga C, et al. Double blind placebo control trial of large neutral amino acids in treatment of PKU: effect on blood phenylalanine. J Inherit Metab Dis. 2007;30(2):153–8. doi: 10.1007/s10545-007-0556-4 [DOI] [PubMed] [Google Scholar]
39.Matalon R, Michals-Matalon K, Bhatia G, Grechanina E, Novikov P, McDonald JD, et al. Large neutral amino acids in the treatment of phenylketonuria (PKU). J Inherit Metab Dis. 2006;29(6):732–8. doi: 10.1007/s10545-006-0395-8 [DOI] [PubMed] [Google Scholar]
40.Moats RA, Moseley KD, Koch R, Nelson M. Brain Phenylalanine Concentrations in Phenylketonuria: Research and Treatment of Adults. Pediatrics. 2003;112(Supplement 4):1575–9. [PubMed] [Google Scholar]
41.van Vliet D, Bruinenberg VM, Mazzola PN, van Faassen M, de Blaauw P, Kema IP, et al. Large Neutral Amino Acid Supplementation Exerts Its Effect through Three Synergistic Mechanisms: Proof of Principle in Phenylketonuria Mice. Plos One. 2015;10(12). doi: 10.1371/journal.pone.0143833 [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Harrison FE, Hosseini AH, McDonald MP. Endogenous anxiety and stress responses in water maze and Barnes maze spatial memory tasks. Behavioural Brain Research. 2009;198(1):247–51. doi: 10.1016/j.bbr.2008.10.015 [DOI] [PMC free article] [PubMed] [Google Scholar]
43.Patil SS, Sunyer B, Höger H, Lubec G. Evaluation of spatial memory of C57BL/6J and CD1 mice in the Barnes maze, the Multiple T-maze and in the Morris water maze. Behavioural Brain Research. 2009;198(1):58–68. doi: 10.1016/j.bbr.2008.10.029 [DOI] [PubMed] [Google Scholar]
44.Christensen R, Alhonen L, Wahlfors J, Jakobsen M, Jensen TG. Characterization of transgenic mice with the expression of phenylalanine hydroxylase and GTP cyclohydrolase I in the skin. Exp Dermatol. 2005;14(7):535–42. doi: 10.1111/j.0906-6705.2005.00326.x [DOI] [PubMed] [Google Scholar]
45.Bruinenberg VM, van der Goot E, van Vliet D, de Groot MJ, Mazzola PN, Heiner-Fokkema MR, et al. The Behavioral Consequence of Phenylketonuria in Mice Depends on the Genetic Background. Frontiers in Behavioral Neuroscience. 2016;10:233. doi: 10.3389/fnbeh.2016.00233 [DOI] [PMC free article] [PubMed] [Google Scholar]
46.van Vliet D, Bruinenberg VM, Mazzola PN, van Faassen MH, de Blaauw P, Pascucci T, et al. Therapeutic brain modulation with targeted large neutral amino acid supplements in the Pah-enu2 phenylketonuria mouse model. Am J Clin Nutr. 2016;104(5):1292–300. doi: 10.3945/ajcn.116.135996 [DOI] [PubMed] [Google Scholar]
47.van Spronsen FJ, Enns GM. Future treatment strategies in phenylketonuria. Mol Genet Metab. 2010;99 Suppl 1:S90–5. doi: 10.1016/j.ymgme.2009.10.008 [DOI] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0261150.r001

Decision Letter 0

Clemens Fürnsinn

26 Feb 2020

PONE-D-19-32961

Effect of offering casein glycomacropeptide versus free synthetic amino acids to early treated phenylketonuria mice

PLOS ONE

Dear Dr. Ahring,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Both reviewers raised some comprehensible matters, which should improve the quality of your paper and render it finally acceptable. Please seriously consider and respond to all these points one by one.

In particular, data from untreated controls would be appreciated for comparison, which can also be via reference to another paper containing this information. With regard to statistics reviewer 1 suggests one-way ANOVA instead of multiple t tests - this could be more convioncing, but multiple t tests are formally correct, so I am inclined to leave the final choice on your side.

Here follows an additional comment from an expert reviewer, which appears meaningful: "There is a long history in the field that simply taking large amounts of large neutral amino acids orally can help protect the brain from damage from elevated phenylalanine. This is all based upon an overly simplistic model of amino acid transport at the blood brain barrier. Investigators continue to work to prove this model even though their own data disprove it and they keep doing statistical manipulations to try to 'prove' their point instead of just taking the data at face value and accepting what the data are showing them which is that in the face of very elevated blood phenylalanine, no amount of amino acid supplementation will really effect brain phenylalanine unless it accomplishes the goal of substantially also lowering the blood phenylalanine." You should at least discuss this critical view about the present state of research in the discussion section.

We would appreciate receiving your revised manuscript by Apr 11 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Clemens Fürnsinn, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

3. Thank you for stating the following in the Financial Disclosure section:

"KKA was an industrial PhD student sponsored by Arlafoodsingredients (AFI) and The Danish Agency for Science, Technology and Innovation and this manuscript is part of the Ph.D. thesis. EJ is employed as food scientist at AFI and contributed with his knowledge about the product GMP."

We note that one or more of the authors have an affiliation to the commercial funders of this research study : 'Arla Foods Ingredients Group P/S, Viby J, Denmark'.

a) Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

Please also include the following statement within your amended Funding Statement.

“The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”

If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.

b) Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc.

Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If this adherence statement is not accurate and there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf.

4. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors report on experiments to compare the effect of adding casein glycomacropeptide (CGMP) to a casein based diet upon the hyperphenylalaninemia phenotype in Pahenu2 mice, basically in an attempt to see if GMP might be work similarly to large neutral amino acid (LNAA) supplements in untreated PKU. They compared the effects to mice treated with dietary protein restriction with or without CGMP supplement. The manuscript is generally well written but there are several typographic errors including a recurring one that says ‘Error! Reference source not found.’

The clarity of the statistical analysis used in the study needs to be improved. Vast numbers of p values are provided, primarily in the supporting information, between many variables, but how these p values were generated is not noted. The statistics sections states the use of t tests and ANOVAs but the results sections never state when these are used. From my reading, five different treatment groups in all female mice were established from the outset. The only viable statistical method then would be a one-way ANOVA to examine the overall effect of treatment across the five groups with a post hoc intergroup comparison thereafter. There would be no place for choosing to employ t tests between multiple pairs of groups as this would lead to compounded errors.

By inspection of the final blood Phe concentrations, I would estimate, if ANOVA were used, a significant treatment effect across all groups given the large difference between the groups on casein and those on Phe restriction; however, it is difficult to estimate whether intergroup differences reach statistical significance between the animals on casein but receiving CGMP or not. More importantly, I doubt intergroup differences in brain Phe, Tyr, or Trp or any of the neurotransmitters are significant at all in the groups that received a casein containing diet.

I recommend that the bar graphs be converted to whisker plots so that the true mean and range of the data can be inspected. This would apply to Figure 3 and supplementary Figure 1.

For Figure 3, I recommend that the amino acids be separated from the neurotransmitters into different charts as the amounts measured differ by a two orders of magnitude and it makes inspection of the current figure difficult.

If it’s possible to find a suitable short phrase or abbreviation, I recommend naming the actual treatment of the groups in the figures rather than using Group 1, group 2, etc in the figures as I found myself having to go back repeatedly to refresh my memory on what each treatment group actually was. I can remember a couple treatments but not five.

As the authors have clearly learned, the lack of either wild type B6 mice or untreated B6-Pahenu2 mice as controls in the behavioral studies make the current results difficult to interpret. Given the decrease in distance traveled, the animals are clearly learning the maze, which I suspect is an improvement over what they would have done had they not been treated from early in life, but do the authors have any maze data on wild type mice or untreated B6-Pahenu2 mice (not collected contemporaneously obviously) to compare to?

The timing of the dietary treatment is bit unclear from the manuscript. It is stated in the manuscript that the pups were products of homozygous Pahenu2 dams treated with Phe-restricted diet and this diet was continued through weaning. Figure 1 shows experimental diets being initiated sometime between week 4-16 and continuing to week 19. When precisely did the experimental diet start? What diet were the mice fed between weaning and the onset of the experimental diet. This would have influenced their ability to perform the maze testing.

Incidentally, the use of the low Phe diet in the dams clearly allows them to generate progeny but since their milk would contain normal concentrations of lactalbumin, I would except Pahenu2 homozygous pups to become hyperphenylalaninemic regardless of whether the dam continued on a low Phe diet or not. Do the authors have blood Phe data on the progeny at weaning or prior to the institution of the experimental diets? The pups may have suffered sufficient brain damage from hyperphenylalaninemia during the juvenile period that Phe lowering treatment instituted later in life may have had little effect on behavior in the animals.

The current concept of LNAA transport at the blood brain barrier being mediated solely via the LAT-1 transporter is exceedingly inadequate. There is evidence for a number of other transporters, some that transport amino acids in reverse direction against the gradient, being involved in brain amino acid homeostasis. The existence of this system is why dietary manipulation in this experiment had little effect upon brain amino acid content other than Phe.

The authors make the statement in their introduction that the imbalance in brain LNAA is ‘probably the primary cause of disrupted brain development in this disorder’ and then cite a single reference. This statement denies abundant evidence and dozens of other publications on a multitude of other potential pathogenic mechanisms; the statement should be eliminated.

The authors find improved bone density primarily in the groups of animals on the Phe-restricted diets yet make no statement about the potential pathogenesis of hyperphenylalaninemia itself upon bone health.

The authors conclude that CGMP can be a ‘relevant supplement for the treatment of PKU.’ I don’t disagree. However, in my opinion, the more important conclusion is that dietary Phe-restriction, potentially using CGMP as part of the Phe-restricted diet, leads to vastly superior outcomes using multiple measures in comparison to ignoring Phe restriction and trying to supplement with LNAA.

Reviewer #2: This is a valuable study, but the separation of Phe intake through water from all other AA in G4 and G5 makes it difficult to compare data. The text is very dry to read and I would recommend to combine results and discussion to give the reader more guidance. Some of the discussion is just a repeat of results. Please also include the supplementary data in the main part of the manuscript.

Page 13 diets: Please explain diet composition of G3.

Page 13 of submitted pdf; Phenylalanine supplementation: 57mg/ml would be an extraordinary amount of Phe (345 mM) and not suitable for the mouse strain. Probably 57mg/L.

Page 19/20: There are two error messages in the text.

Table 2: Threonine is elevated in G4 and G5. It is an essential AA and diet 5 has a significant amount, but diet 4 doesn't seem to have Thr. Please explain. Arginine is consistent and found in G4 and G5 diets.

I would recommend to convert table 2 into a bar graph.

Figures in the pdf are of unacceptable resolution.

Fig 3 Please present control data from a background mouse strain. The figure caption needs to explain the information content of the right panel. Do not abbreviate brain areas.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Cary O. Harding

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2022 Jan 11;17(1):e0261150. doi: 10.1371/journal.pone.0261150.r002

Author response to Decision Letter 0

27 Sep 2020

Once again, we thank you for the time you put in reviewing our paper and look forward to meeting your expectations. Since your inputs have been precious, in the eventuality of a publication, we would like to acknowledge your contribution explicitly.

Attachment

Submitted filename: Response to reviewers.rtf

Click here for additional data file.^{(77.3KB, rtf)}

PLoS One. doi: 10.1371/journal.pone.0261150.r003

Decision Letter 1

Clemens Fürnsinn

8 Oct 2020

PONE-D-19-32961R1

Effect of offering casein glycomacropeptide versus free synthetic amino acids to early treated phenylketonuria mice

PLOS ONE

Dear Dr. Ahring,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we have decided that your manuscript does not meet our criteria for publication and must therefore be rejected.

It was the reviewer´s feeling that the extent of changes and re-writing of the manuscript was not sufficient (and that there was not a substantiated rebuttal outlining why this was so). I must say that I share the impression that several points remained unclarified and that the changes were less extensive than suggested by the reviewers. Your response to the reviewers is rather brief and falls back behind the point-by-point explanations that we are used to receive with detailed elaboration and reasoning of which changes have been made (or not made), and why.

I am sorry that we cannot be more positive on this occasion, but hope that you appreciate the reasons for this decision.

Yours sincerely,

Clemens Fürnsinn, Ph.D.

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #2: No

**********

6. Review Comments to the Author

Reviewer #2: The study requires a significant rewrite. Results and Discussion should be combined. The concentration of phenylalanine given to the animals does not have correct units.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

- - - - -

For journal use only: PONEDEC3

PLoS One. 2022 Jan 11;17(1):e0261150. doi: 10.1371/journal.pone.0261150.r004

Author response to Decision Letter 1

30 Apr 2021

To whom it may concern

My colleagues and I humbly re-submit our manuscript entitled “The effect of Casein glycomacropeptide versus free synthetic amino acids for early treatment of phenylketonuria in a mice model” for your re-consideration.

We wish to thank you all for your constructive comments in this second round of review. Your comments provided valuable insights to refine its contents and analysis. In this document, we try to address the issues raised as best as possible

Authors respond to reviewers PLOS ONE answers

(Reviewer #1) Thank you very much for your suggestion regarding using one-way ANOVA instead of multiple t tests. During my PhD study period, I did seek advice at the Statistical Department at the University of Copenhagen and the teacher recommended that I should use multiple t-tests in order to obtain the most transparent results. He stated, that in his opinion it can confuse the outcome to include to many parameters in the same statistical analysis. Therefore, I choose to keep this method as kindly offered from the Academic Editor.

I recommend that the bar graphs be converted to whisker plots so that the true mean and range of the data can be inspected. This would apply to Figure 3 and supplementary Figure 1.

Thank you for the suggestion, we have converted the bar graphs Figure 3 and supplementary Figure 1 to whisker plots.

For Figure 3, I recommend that the amino acids be separated from the neurotransmitters into different charts as the amounts measured differ by two orders of magnitude and it makes inspection of the current figure difficult.

Thank you for the recommendation for Figure 3, we have separated the amino acids from the neurotransmitters into different charts to make the inspection of the current figure easier.

We do agree that a suitable short phrase or abbreviation would be preferable for naming the actual treatment of the groups. We have done as follows: Re-named the 5 groups and added this information to glossary.

Thank you for addressing this very important issue regarding lack of wild type mice. We did not have a wild type mouse to compare to, which we initial found was not a problem, since the main objective of the study was to determine the differences between the diet compositions. We are aware that similar studies has used wild type mouse to compare with ( (Ney, 2008) (Vliet, 2016) and we have realized afterwards, that this could have been useful for the Barnes maze study, since there were no differences between the groups, despite the different diet regimes. We have included a comment about this in the discussion section.

The experimental diet started at week 4, right after weaning was ended. The breeding animals were maintained on Phe-free semi synthetic diet (Harlan Laboratories), also during pregnancy and weaning period, a Maternal PKU diet. As the diet was free of Phe, the drinking water was supplemented with Phe (Sigma-Aldrich Chemie) to a final concentration of 62.5 mg/ml during that period.

We have now included a new figure 1 to make it clearer.

Yes we agree that this could be a problem however It was not possible to take a blood test for determination of the total amino acid profile at week 1 of diet intervention since the mice at that time, were too small to tolerate blood sampling, but as all animals were treated equal before week 1, we assume that the start value was similar in all groups.

We have removed the sentence as requested by reviewer.

Thank you for pointing out that statement about the potential pathogenesis of hyperphenylalaninemia upon bone health was missing. We have in the discussion included the impact on bone density.

The authors conclude that CGMP can be a ‘relevant supplement for the treatment of PKU.’ I don’t disagree. However, in my opinion, the more important conclusion is that dietary Phe-restriction, potentially using CGMP as part of the Phe-restricted diet, leads to vastly superior outcomes using multiple measures in comparison to ignore Phe restriction and trying to supplement with LNAA

Also thank you for emphasizing, that it is important to highlight the fact, that taking large amounts of large neutral amino acids orally can help protect the brain from damage from elevated phenylalanine, but the main lowering effect on brain phenylalanine comes from lowering the blood phenylalanine. We thought we already indicated that but have now expanded the discussion section where we describe in more detail with the work of others, as you advised us to do.

Reviewer 2#

This is a valuable study, but the separation of Phe intake through water from all other AA in G4 and G5 makes it difficult to compare data. The text is very dry to read and I would recommend to combine results and discussion to give the reader more guidance. Some of the discussion is just a repeat of results. Please also include the supplementary data in the main part of the manuscript.

Thank you for your valuable advices regarding design of the manuscript. We do agree that the text can be dry to read, and therefore we have combined “results-“and “discussion sections” a bit more as suggested and included the supplementary data in the main part of the manuscript.

Page 13 diets: Please explain diet composition of G3.

The composition of G3 is a complete diet, consisting of casein (MIPRODAN® 30), sucrose, corn starch, corn oil, cellulose, mineral- and vitamin-mix. Basically, the nutritional value is the same as the other experimental groups. We have now included a detailed description of G3 in Table 2

Page 13 of submitted pdf; Phenylalanine supplementation: 57mg/ml would be an extraordinary amount of Phe (345 mM) and not suitable for the mouse strain. Probably 57mg/L.

Thank you for pointing out that 57mg/ml of Phenylalanine supplementation would be an extraordinary amount of Phe (345 mM) and not suitable for the mouse strain. In fact, 25 mg/ml was used to produce the drinking mixture, where 9 ml (225 mg) was added to 391 ml of water, which provided a final concentration of 563 mg/l

Page 19/20: There are two error messages in the text.

Thank you for making us aware of this, it is corrected now.

We are glad that you pointed this out, since it is not clear at all from the table 2, that the CGMP product contains large amount of Thr. We have now added the data from CGMP-20 to make it more obvious

I would recommend to convert table 2 into a bar graph. Figures in the pdf are of unacceptable resolution.

We hope the resolution of the figures have improved with the new system: uploading figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool https://pacev2.apexcovantage.com/

Fig 3 Please present control data from a background mouse strain. The figure caption needs to explain the information content of the right panel.

Unfortunately, the mouse strain we used from AAU had no wild type as described in the introduction section, but we have found useful information from other similar studies (Ney, 2008) (Vliet, 2016)

We were aware from the start of the study, that a wild type of the mouse strain would have been a useful tool. However, the information we needed from the study would be possible to gain, also without a wild type, but that fact determined the design of the study: 2 groups with pure GCMP, 1 group on normal diet, 2 groups with FSAA

We expected to find a difference between the groups due to the fact, that late-diagnosed/untreated PKU patients over the years have been studied and observed.

Do not abbreviate brain areas.

We have now changes to full names for brain areas

Attachment

Submitted filename: Response to reviewers.rtf

Click here for additional data file.^{(143.3KB, rtf)}

PLoS One. doi: 10.1371/journal.pone.0261150.r005

Decision Letter 2

Aneta Agnieszka Koronowicz

25 Nov 2021

The effect of Casein glycomacropeptide versus free synthetic amino acids for early treatment of phenylketonuria in a mice model

PONE-D-19-32961R2

Dear Dr. ahring,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Aneta Agnieszka Koronowicz, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The Authors introduce discussion elements into the results section and vice versa, so it is recommended to combine the results and the discussion (Results and Discussion).

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #2: The concerns raised by this reviewer were addressed. I still would recommend to combine results and discussion.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

PLoS One. doi: 10.1371/journal.pone.0261150.r006

Acceptance letter

Aneta Agnieszka Koronowicz

7 Dec 2021

PONE-D-19-32961R2

The effect of Casein glycomacropeptide versus free synthetic amino acids for early treatment of phenylketonuria in a mice model

Dear Dr. Ahring:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Prof. Aneta Agnieszka Koronowicz

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Attachment

Submitted filename: Response to reviewers.rtf

Click here for additional data file.^{(77.3KB, rtf)}

Attachment

Submitted filename: Response to reviewers.rtf

Click here for additional data file.^{(143.3KB, rtf)}

Data Availability Statement

All relevant data are within the paper.

[pone.0261150.ref001] 1.Blau N, Hennermann JB, Langenbeck U, Lichter-Konecki U. Diagnosis, classification, and genetics of phenylketonuria and tetrahydrobiopterin (BH4) deficiencies. Mol Genet Metab. 2011;104 Suppl:S2–9. [DOI] [PubMed] [Google Scholar]

[pone.0261150.ref002] 2.Blau N, van Spronsen FJ, Levy HL. Phenylketonuria. Lancet. 2010;376(9750):1417–27. doi: 10.1016/S0140-6736(10)60961-0 [DOI] [PubMed] [Google Scholar]

[pone.0261150.ref003] 3.Mitchell JJ, Trakadis YJ, Scriver CR. Phenylalanine hydroxylase deficiency. Genet Med. 2011;13(8):697–707. doi: 10.1097/GIM.0b013e3182141b48 [DOI] [PubMed] [Google Scholar]

[pone.0261150.ref004] 4.Schuck PF, Malgarin F, Cararo JH, Cardoso F, Streck EL, Ferreira GC. Phenylketonuria Pathophysiology: on the Role of Metabolic Alterations. Aging Dis. 2015;6(5):390–9. doi: 10.14336/AD.2015.0827 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261150.ref005] 5.van Wegberg AMJ, MacDonald A, Ahring K, Bélanger-Quintana A, Blau N, Bosch AM, et al. The complete European guidelines on phenylketonuria: diagnosis and treatment. Orphanet journal of rare diseases. 2017;12(1):162-. doi: 10.1186/s13023-017-0685-2 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261150.ref006] 6.Demirdas S, Coakley KE, Bisschop PH, Hollak CEM, Bosch AM, Singh RH. Bone health in phenylketonuria: a systematic review and meta-analysis. Orphanet Journal of Rare Diseases. 2015;10(1):17. doi: 10.1186/s13023-015-0232-y [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261150.ref007] 7.MacDonald A, Lilburn M, Davies P, Evans S, Daly A, Hall SK, et al. ’Ready to drink’ protein substitute is easier is for people with phenylketonuria. J Inherit Metab Dis. 2006;29(4):526–31. doi: 10.1007/s10545-006-0234-y [DOI] [PubMed] [Google Scholar]

[pone.0261150.ref008] 8.Giovannini M, Verduci E, Salvatici E, Paci S, Riva E. Phenylketonuria: nutritional advances and challenges. Nutr Metab (Lond). 2012;9(1):7. doi: 10.1186/1743-7075-9-7 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261150.ref009] 9.Zimmermann M, Jacobs P, Fingerhut R, Torresani T, Thöny B, Blau N, et al. Positive effect of a simplified diet on blood phenylalanine control in different phenylketonuria variants, characterized by newborn BH4 loading test and PAH analysis. Mol Genet Metab. 2012;106(3):264–8. doi: 10.1016/j.ymgme.2012.04.016 [DOI] [PubMed] [Google Scholar]

[pone.0261150.ref010] 10.Aimutis WR. Bioactive properties of milk proteins with particular focus on anticariogenesis. J Nutr. 2004;134(4):989s–95s. doi: 10.1093/jn/134.4.989S [DOI] [PubMed] [Google Scholar]

[pone.0261150.ref011] 11.Etzel MR. Manufacture and use of dairy protein fractions. The Journal of Nutrition. 2004;134(4):996S. doi: 10.1093/jn/134.4.996S [DOI] [PubMed] [Google Scholar]

[pone.0261150.ref012] 12.Ney DM, Hull AK, van Calcar SC, Liu X, Etzel MR. Dietary glycomacropeptide supports growth and reduces the concentrations of phenylalanine in plasma and brain in a murine model of phenylketonuria. J Nutr. 2008;138(2):316–22. doi: 10.1093/jn/138.2.316 [DOI] [PubMed] [Google Scholar]

[pone.0261150.ref013] 13.Van Calcar SC, Hull AK, Liu X, Etzel M, Ney DM. Dietary glycomacropeptide (GMP) supports growth and reduces the concentrations of phenylalanine in plasma and brain in the PKU mouse. Mol Genet Metab. 2008;93(3):263-. [DOI] [PubMed] [Google Scholar]

[pone.0261150.ref014] 14.van Calcar SC, MacLeod EL, Gleason ST, Etzel MR, Clayton MK, Wolff JA, et al. Improved nutritional management of phenylketonuria by using a diet containing glycomacropeptide compared with amino acids. Am J Clin Nutr. 2009;89(4):1068–77. doi: 10.3945/ajcn.2008.27280 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261150.ref015] 15.van Calcar SC, Macleod EL, Gleason ST, Etzel MR, Rice GM, Ney DM. Glycomacropeptide (GMP): A new option for PKU diet management. Mol Genet Metab. 2011;102(3):262-. [Google Scholar]

[pone.0261150.ref016] 16.van Calcar SC, Ney DM. Food Products Made with Glycomacropeptide, a Low-Phenylalanine Whey Protein, Provide a New Alternative to Amino Acid-Based Medical Foods for Nutrition Management of Phenylketonuria. J Acad Nutr Diet. 2012;112(8):1201–10. doi: 10.1016/j.jand.2012.05.004 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261150.ref017] 17.Zaki OK, El-Wakeel L, Ebeid Y, Elarab HSE, Moustafa A, Abdulazim N, et al. The Use of Glycomacropeptide in Dietary Management of Phenylketonuria. Journal of Nutrition and Metabolism. 2016. doi: 10.1155/2016/2453027 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261150.ref018] 18.Schindeler S, Ghosh-Jerath S, Thompson S, Rocca A, Joy P, Kemp A, et al. The effects of large neutral amino acid supplements in PKU: an MRS and neuropsychological study. Mol Genet Metab. 2007;91(1):48–54. doi: 10.1016/j.ymgme.2007.02.002 [DOI] [PubMed] [Google Scholar]

[pone.0261150.ref019] 19.Rocha JC, Martel F. Large neutral amino acids supplementation in phenylketonuric patients. J Inherit Metab Dis. 2009;32(4):472–80. doi: 10.1007/s10545-009-1132-x [DOI] [PubMed] [Google Scholar]

[pone.0261150.ref020] 20.Lou HC, Güttler F, Lykkelund C, Bruhn P, Niederwieser A. Decreased vigilance and neurotransmitter synthesis after discontinuation of dietary treatment for phenylketonuria in adolescents. Eur J Pediatr. 1985;144(1):17–20. doi: 10.1007/BF00491918 [DOI] [PubMed] [Google Scholar]

[pone.0261150.ref021] 21.Lou HC, Lykkelund C, Gerdes AM, Udesen H, Bruhn P. Increased vigilance and dopamine synthesis by large doses of tyrosine or phenylalanine restriction in phenylketonuria. Acta Paediatr Scand. 1987;76(4):560–5. doi: 10.1111/j.1651-2227.1987.tb10521.x [DOI] [PubMed] [Google Scholar]

[pone.0261150.ref022] 22.Pascucci T, Giacovazzo G, Andolina D, Accoto A, Fiori E, Ventura R, et al. Behavioral and neurochemical characterization of new mouse model of hyperphenylalaninemia. PLoS One. 2013;8(12):e84697. doi: 10.1371/journal.pone.0084697 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261150.ref023] 23.Shedlovsky A, McDonald JD, Symula D, Dove WF. Mouse models of human phenylketonuria. Genetics. 1993;134(4):1205–10. doi: 10.1093/genetics/134.4.1205 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261150.ref024] 24.Mekada K, Abe K, Murakami A, Nakamura S, Nakata H, Moriwaki K, et al. Genetic differences among C57BL/6 substrains. Exp Anim. 2009;58(2):141–9. doi: 10.1538/expanim.58.141 [DOI] [PubMed] [Google Scholar]

[pone.0261150.ref025] 25.Ahring K, Kjolby M. Effect of offering Phenylketonuria (PKU) mice Glycomacropeptide (GMP) before normal or low protein (LP) diet on growth, serum-phenylalanine (phe)-concentration, bone structure, performance and concentration of phe in the brain. protocols.io doi: 10.17504/protocols.io.bkg9ktz6 [DOI] [Google Scholar]

[pone.0261150.ref026] 26.Benevenga NJ, Gahl MJ, Crenshaw TD, Finke MD. Protein and amino acid requirements for maintenance and amino acid requirements for growth of laboratory rats. J Nutr. 1994;124(3):451–3. doi: 10.1093/jn/124.3.451 [DOI] [PubMed] [Google Scholar]

[pone.0261150.ref027] 27.Johannik K, Vanhecke P, Francois B, Marchal G, Smet MH, Jaeken J, et al. Localized Brain Proton Nmr-Spectroscopy in Young-Adult Phenylketonuria Patients. Magn Reson Med. 1994;31(1):53–7. [DOI] [PubMed] [Google Scholar]

[pone.0261150.ref028] 28.Protein and amino acid requirements in human nutrition. World Health Organ Tech Rep Ser. 2007(935):1–265, back cover. [PubMed] [Google Scholar]

[pone.0261150.ref029] 29.Peake RW, Law T, Hoover PN, Gaewsky L, Shkreta A, Kellogg MD. Improved separation and analysis of plasma amino acids by modification of the MassTrak AAA Solution Ultraperformance(R) liquid chromatography method. Clin Chim Acta. 2013;423:75–82. doi: 10.1016/j.cca.2013.03.036 [DOI] [PubMed] [Google Scholar]

[pone.0261150.ref030] 30.Pritchett K, Mulder GB. T, radial arm, and Barnes mazes. Contemp Top Lab Anim Sci. 2003;42(3):53–5. [PubMed] [Google Scholar]

[pone.0261150.ref031] 31.Miller DJ. Sydney Ringer; physiological saline, calcium and the contraction of the heart. The Journal of Physiology. 2004;555(Pt 3):585–7. doi: 10.1113/jphysiol.2004.060731 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261150.ref032] 32.Solverson P, Murali SG, Litscher SJ, Blank RD, Ney DM. Low bone strength is a manifestation of phenylketonuria in mice and is attenuated by a glycomacropeptide diet. PloS one. 2012;7(9):e45165–e. doi: 10.1371/journal.pone.0045165 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261150.ref033] 33.Berry HK, Brunner RL, Hunt MM, White PP. Valine, isoleucine, and leucine. A new treatment for phenylketonuria. Am J Dis Child. 1990;144(5):539–43. doi: 10.1001/archpedi.1990.02150290033020 [DOI] [PubMed] [Google Scholar]

[pone.0261150.ref034] 34.Pascucci T, Andolina D, Ventura R, Puglisi-Allegra S, Cabib S. Reduced availability of brain amines during critical phases of postnatal development in a genetic mouse model of cognitive delay. Brain Res. 2008;1217:232–8. doi: 10.1016/j.brainres.2008.04.006 [DOI] [PubMed] [Google Scholar]

[pone.0261150.ref035] 35.Harding CO, Winn SR, Gibson KM, Arning E, Bottiglieri T, Grompe M. Pharmacologic inhibition of L-tyrosine degradation ameliorates cerebral dopamine deficiency in murine phenylketonuria (PKU). J Inherit Metab Dis. 2014;37(5):735–43. doi: 10.1007/s10545-013-9675-2 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261150.ref036] 36.Nakano T, Silva-Hernandez ER, Ikawa N, Ozimek L. Purification of kappa-casien glycomacropeptide from sweet whey with undetectable level of phenylalanine. Biotechnol Prog. 2002;18(2):409–12. doi: 10.1021/bp010195u [DOI] [PubMed] [Google Scholar]

[pone.0261150.ref037] 37.Puglisi-Allegra S, Cabib S, Pascucci T, Ventura R, Cali F, Romano V. Dramatic brain aminergic deficit in a genetic mouse model of phenylketonuria. Neuroreport. 2000;11(6):1361–4. doi: 10.1097/00001756-200004270-00042 [DOI] [PubMed] [Google Scholar]

[pone.0261150.ref038] 38.Matalon R, Michals-Matalon K, Bhatia G, Burlina AB, Burlina AP, Braga C, et al. Double blind placebo control trial of large neutral amino acids in treatment of PKU: effect on blood phenylalanine. J Inherit Metab Dis. 2007;30(2):153–8. doi: 10.1007/s10545-007-0556-4 [DOI] [PubMed] [Google Scholar]

[pone.0261150.ref039] 39.Matalon R, Michals-Matalon K, Bhatia G, Grechanina E, Novikov P, McDonald JD, et al. Large neutral amino acids in the treatment of phenylketonuria (PKU). J Inherit Metab Dis. 2006;29(6):732–8. doi: 10.1007/s10545-006-0395-8 [DOI] [PubMed] [Google Scholar]

[pone.0261150.ref040] 40.Moats RA, Moseley KD, Koch R, Nelson M. Brain Phenylalanine Concentrations in Phenylketonuria: Research and Treatment of Adults. Pediatrics. 2003;112(Supplement 4):1575–9. [PubMed] [Google Scholar]

[pone.0261150.ref041] 41.van Vliet D, Bruinenberg VM, Mazzola PN, van Faassen M, de Blaauw P, Kema IP, et al. Large Neutral Amino Acid Supplementation Exerts Its Effect through Three Synergistic Mechanisms: Proof of Principle in Phenylketonuria Mice. Plos One. 2015;10(12). doi: 10.1371/journal.pone.0143833 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261150.ref042] 42.Harrison FE, Hosseini AH, McDonald MP. Endogenous anxiety and stress responses in water maze and Barnes maze spatial memory tasks. Behavioural Brain Research. 2009;198(1):247–51. doi: 10.1016/j.bbr.2008.10.015 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261150.ref043] 43.Patil SS, Sunyer B, Höger H, Lubec G. Evaluation of spatial memory of C57BL/6J and CD1 mice in the Barnes maze, the Multiple T-maze and in the Morris water maze. Behavioural Brain Research. 2009;198(1):58–68. doi: 10.1016/j.bbr.2008.10.029 [DOI] [PubMed] [Google Scholar]

[pone.0261150.ref044] 44.Christensen R, Alhonen L, Wahlfors J, Jakobsen M, Jensen TG. Characterization of transgenic mice with the expression of phenylalanine hydroxylase and GTP cyclohydrolase I in the skin. Exp Dermatol. 2005;14(7):535–42. doi: 10.1111/j.0906-6705.2005.00326.x [DOI] [PubMed] [Google Scholar]

[pone.0261150.ref045] 45.Bruinenberg VM, van der Goot E, van Vliet D, de Groot MJ, Mazzola PN, Heiner-Fokkema MR, et al. The Behavioral Consequence of Phenylketonuria in Mice Depends on the Genetic Background. Frontiers in Behavioral Neuroscience. 2016;10:233. doi: 10.3389/fnbeh.2016.00233 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261150.ref046] 46.van Vliet D, Bruinenberg VM, Mazzola PN, van Faassen MH, de Blaauw P, Pascucci T, et al. Therapeutic brain modulation with targeted large neutral amino acid supplements in the Pah-enu2 phenylketonuria mouse model. Am J Clin Nutr. 2016;104(5):1292–300. doi: 10.3945/ajcn.116.135996 [DOI] [PubMed] [Google Scholar]

[pone.0261150.ref047] 47.van Spronsen FJ, Enns GM. Future treatment strategies in phenylketonuria. Mol Genet Metab. 2010;99 Suppl 1:S90–5. doi: 10.1016/j.ymgme.2009.10.008 [DOI] [PubMed] [Google Scholar]

PERMALINK

The effect of casein glycomacropeptide versus free synthetic amino acids for early treatment of phenylketonuria in a mice model

Kirsten K Ahring

Frederik Dagnæs-Hansen

Annemarie Brüel

Mette Christensen

Erik Jensen

Thomas G Jensen

Mogens Johannsen

Karen S Johansen

Allan M Lund

Jesper G Madsen

Karen Brøndum-Nielsen

Michael Pedersen

Lambert K Sørensen

Mads Kjolby

Lisbeth B Møller

Roles

Abstract

Introduction

Objective

Material and methods

Results

Conclusion

Introduction

Material and methods

Animals

The research diets

Table 1. Composition of the experimental diet for the 5 groups (G1, G2, G3, G4, G5) and content of AA in CGMP-20 (g AA/100 g protein).

Table 2. Analysis of diets G1-G5.

Weekly Phe and Tyr analyses

AA profile analysis in plasma

Body composition analysis

Behavior performance test (Barne’s maze behavioral test)

Brain and bone removal

Analysis of Phe, Tyr, Trp, neurotransmitters, and their metabolites in the brain regions

Bone examination

Statistical analysis

The experimental design

Fig 1. Timeline of the study design (weeks refer to the week of diet treatment, which continuous from week 1 until week 15.

Results

Food and water

Fig 2.

Growth (weight start, weight end, weight gain) and body scan (fat, lean and weight)

Bone density measurements/bone examination

Weekly Phe and Tyr plasma analyses (Dried Blood Spot (DBS)

Fig 3. Average plasma Phe, plasma Tyr- and plasma Phe/Tyr-ratio (μmol/l), mean ± SD.

Total AA profile (week 6 and 12 of the diet intervention period)

Fig 4. Average plasma concentrations of AA at w6 and w12.

Table 3. P-values for comparison of the different groups G1-G5, representing G1(N-CGMP), G2 (N-CGMP-LNAA), G3 (N), G4 (CGMP-EAA-LP) and G5 (FSAA-LP).

Table 4. Plasma content of selected AA in plasma measured at week 6 and 12 presented as mean and SD, difference from start to end and % change.

Brain examination

Fig 5. The total content of DA, 5-HT, 5-HIAA, DOPAC, Phe, Tyr and Trp from the following brain parts cerebellum, brain stem, hypothalamus, parietal cortex, anterior piriform, cortex and olfactory bulb (presented as total brain by ading values for the individual brain parts together).

Fig 6. The content of DA, 5-HT, 5-HIAA, DOPAC, Phe, Tyr and Trp in the 5 groups of animals measured separately in the different brain parts; cerebellum, brain stem, hypothalamus, parietal cortex, anterior piriform, cortex and olfactory bulb.

Table 5. Difference in concentrations of selected AA and metabolites in different brain parts.

Fig 7. Correlation of the content of Phe, Tyr, Trp (plasma and brain) and the neurotransmitters, 5-HT, DA, and their metabolites 5-HIAA, DOPAC in the total brain.

Behavioral test (Barnes maze)

Fig 8. Barnes maze.

Exclusion of animals with malocclusion due to incisor overgrowth

Discussion

Conclusion

Acknowledgments

Glossary

Data Availability

Funding Statement

References

Decision Letter 0

Clemens Fürnsinn

Roles

Author response to Decision Letter 0

Decision Letter 1

Clemens Fürnsinn

Roles

Author response to Decision Letter 1

Decision Letter 2

Aneta Agnieszka Koronowicz

Roles

Acceptance letter

Aneta Agnieszka Koronowicz

Roles